The inactivation of microbial enzymes in vivo.

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THE INACTIVATION OF

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Annu. Rev. Microbiol. 1977.31:135-154. Downloaded from www.annualreviews.org Access provided by Georgetown University on 02/02/15. For personal use only.

MICROBIAL ENZYMES IN VIVO Robert L. Switzer Department of Biochemistry, University of Illinois, Urbana, Illinois 61801

CONTENTS INTRODUCTION ....... ......... ..,........................................................................................... DEFINITIONS AND SCOPE OF THIS REVIEW ...................... ...................... .................. EXAMPLES OF THE INACTIVATION OF MICROBIAL ENZYMES IN VIVO .. ... .. ......... PHYSIOLOGICAL CONDITIONS LEADING TO ENZYME INACTIVATION..................

Shifts in Carbon and Nitrogen Metabolism .............................................................. Differentiation ..................... ....................................................................................... Why Inactivation? ........ . ........................................................ ... ...... ............................ MECHANISMS OF INACTIVATION ................................................................................

Physical Mechanisms.................................................................................................. Chemical Mech anisms................................................................................................ Degradative Inactivation: The Role of Proteolysis and Turnover.............................. Unknown Mechanisms............................�................................................................... METHODS FOR STUDYING INACTIVATION PROCESSES ............................................

General Characterization............................................................................................ Effects of Inhibitors.................................................................................................... Use of Mutant Strains................................................................................................ Reactivation In Vivo and In Vitro ............................ .......... ........................... ........... Following the Fate of the Enzyme Protein During Inactivation . ... ... .. ... .. . .... . .......... Reconstruction In Vitro.............................................................................................. CONCLUDING COMMENTS ......................................................................................... ..

135 136 137 137 142

143

144 144 144 145

145

147

147 147

149 149

151 151 153 154

INTRODUCTION

. Two mechanisms for the regulation of enzyme activity in microbes have been generally recognized and extensively studied. These are control of enzyme levels through regulation of the rate of enzyme synthesis and control of enzyme activity through the effects of noncovalent binding of ligands. In recent years it has become appreciated that another mode of enzyme regulation is generally found in microbes, namely, the control of enzyme activity by selective inactivation. Early experiments 135


136

SWITZER

demonstrating that most bacterial proteins are completely stable in exponentially growing cells, even in the absence of inducing metabolites (5 1, 67, 83, 106), gave rise to the widespread view that the inactivation of enzymes either did not occur in microbes or was of minor importance. However, a considerable body of experi mental evidence has accumulated, which documents the occurrence of selective protein turnover in microbes (reviewed in 36, 37, 95). Reversible covalent modifica tion of enzymes has been shown to serve as an important control mechanism in bacteria and fungi (54, 120). There are numerous reports in the literature of selective inactivation-often by as yet unknown mechanisms-of microbial enzymes under specific physiological conditions. It is the purpose of this review to collect and critically analyze these reports. To my knowledge this subject has not been reviewed previously, although Thurston (129) and Holzer (52) have discussed aspects of the subject in short articles. We will be led to the conclusion that enzyme inactivation is a quite general regulatory mechansim. The role of enzyme inactivation in the physiology of microbes will be considered. The known mechanisms of enzyme inactivation and generally useful methods for studying the mechansim of inactiva tion processes will be examined. DEFINITIONS AND SCOPE OF THIS REVIEW

For the purposes of this review, inactivation will be defined as the irreversible loss in vivo of catalytic activity in the physiologically significant reaction of an enzyme. I have considered only cases where there was clear evidence that the inactivation occurred under physiological conditions in the living cell; inactivation processes that have been studied only in vitro were not reviewed. The term "irreversible loss" deserves further comment. The intention is to distinguish between inactivation and inhibition through the relatively loose, noncovalent binding of inhibitory metabo lites. It is presumed that the latter is readily reversible by dilution or dialysis of cell extracts but that a true inactivation is not reversed by such treatments. I Inactivation processes reversed in vivo by a shift in the metabolism of the cell, such as occurs in the case of reversible covalent modification of enzymes, are included in the general class. The term inactivation is intended to include both modification inactivation, in which the enzyme protein remains intact but loses activity either through a change in physical state or the attachment of a covalent modifying group, and degradative inactivation, in which at least one peptide bond of the protein is cleaved as part of the inactivation process or subsequently to it. Degradative inactivation is taken to be distinct from turnover, which is defined as cleavage of a protein to the constituent amino acids, the more complex process in which degradative inac tivation could serve as the first step. I It

must be conceded that the inactivation of yeast ornithine transcarbamylase by arginase,

which has been included in this review, would have been excluded by this criterion. In this case the inactivation after addition of arginine could be detected when cells were rendered permeable by treatment with nystatin but not in cell-free extracts (141). The inactivation could be reconstructed in vitro if sufficiently high protein concentrations were used.


INACTIVATION OF ENZYMES IN VIVO

137

Since the product of a modification inactivation sometimes leads to the formation of an enzyme that still has catalytic activity [as in the y-glutamyl transferase activity of adenylylated glutamine synthetase of Escherichia coli (1 20)], there is the restric tion that the physiologically significant reaction of the enzyme must be totally (operationally> 95%) lost. Since it often is difficult to ascertain whether a given activity is totally lost or whether some enzyme remains in the unmodified state, this quantitative restriction has not been rigorously applied in this review. Enzyme modification processes in which the physiologically significant reaction is altered but not lost [e.g. modification of fructose bisphosphate aldolase in Bacillus cereus (107) or interconversion of glycogen phosphorylase (27, 35, 128) and glycogen synthetase (55) by phosphorylation and dephosphorylation in eukaryotic microbes] are not included as inactivation processes. It is intended in this review to discuss instances of the inactivation of enzymes, defined as above, that occur in prokaryotic and eukaryotic microbes. Many casual reports of the loss of enzyme activity from microbial cells exist, but I have attempted to concentrate on those cases in which the inactivation process itself was a focus of attention. Cases of the inactivation of enzymes in phage-infected bacteria (29, 47, 8 1 , 99, 109, 1 3 1) and in differentiating slime molds (2, 43, 64, 125) have not been included because of space limitations and because of their specialized nature. EXAMPLES OF THE INACTIVATION OF MICROBIAL ENZYMES IN VIVO

Table 1 lists 41 examples of reasonably well-characterized inactivation processes occurring in microbes that were found in a survey of the literature from about 1945 to mid 1976. It is possible that a few examples belonging in this list have escaped my attention, for which I apologize, but the number and variety of cases in Table 1 serves to document the generality of enzyme inactivation. It is likely that these examples represent only a small fraction of the actual number of inactivation pro cesses that remain to be discovered. PHYSIOLOGICAL CONDITIONS LEADING TO ENZYME INACTIVATION

Enzyme content, as measured by assay of specific catalytic activity in crude extracts, generally remains constant during exponential growth of microbial cells in batch culture. This constancy usually represents a uniform rate of synthesis rather than a balance between synthesis and inactivation, although a low level of protein turn over is known to occur in exponentially growing bacteria (36, 37, 67, 95, 143). Even when the inducing metabolite is removed, most inducible enzymes are stable in exponentially growing cells (51, 97). These observations have tended to obscure the significance of enzyme inactivation as a regulatory mechanism in microbes. But it should be recognized that exponential growth under constant culture conditions would hardly be expected to initiate major changes in metabolic fluxes. Such condi-


Table

1 Examples of the inactivation of microbial enzymes in vivo

...... w 00

Reactivation Enzyme inactivated Aminoacyl-tRNA

Organism(s) Escherchia coli

synthetases

I) -Aminolevulinic acid synthetase

Culture conditions

Proposed mechanism

in vivo without

leading to inactiva tion

of inactivation

protein synthesis

stationary phase owing to

unknown

transfer of derepressed

unknown

amino acid starvation

Rhodopseudomonas spheroides

no

Ref.

144 11,70, 7 1

cells to highly aerobic conditions,addition

of

I) -aminolevulinate

Aspartate

Bacillus subtilis

transcarbamylase

stationary phase, starvation

unknown; energy dependent; cross-reactive protein dis-

no

7,16,126, 138

appears Aspartokinase

B. licheniformis

stationary phase, starvation

unknown

5,42,121

for carbon Aspartokinase,

E. coli

lysine sensitive

stationary phase

unknown; cross-reacting adenylyla tion

Aspartokinase

89a, l 2 7a

protein disappears; Myxococcus xanthus

glycerol-induced myxospore

reported

unknown

105

formation

Citrate lyase

R. geiatinosa

exhaustion of citrate during anaerobic growth in light

Diaminopimeiate

B. cereus

stationary phase

Endonuclease I

E.coli

end of exponential growth

decarboxylase

enzymic removal of acetyl group from a sulfhydryl

group on the enzyme unknown

yes, by enzymic and

6,32-34;40

(in vitro) chemical

'acetyla tion

?

unknown

41 115

on certain media Fructose bisphosphatase Galactozymase

Saccharomyces and Candida spp. S. cerevisiae

addition of glucose to acetate-grown cells addition of glucose to or shift to aerobiosis of galactose grown cells

unknown; proteolysis demonstrated in vitro

unknown; probably energy dependent '

no

30, 86, 136 119

til

� ....

>-! N

�

Table

1 (Continued)


/i-l,3-Glucanase (extracellular)

Basidiomycete QM806

addition of glucose to derepressed cells

unknown; involves extracel-

no

28

lular inactivating system and protein synthesis

Glucose-6-phos-

Bacillus cereus

phate dehydro-

germination and outgrowth of endospores

genase Glucose transport

Neurospora crassa S. cerevisiae, stellatoidea tumefaciens

C. utilis,

Glutamate dehydrogenase,

S. cerevisiae

NAD dependent

dehydrogenase,

Aspergillus

NADP dependent

niduians

C. utilis

Glutamine

synthetase

synthetase

3,8,39,104

suspension in absence of

unknown

transfer to C- and N-free

unknown

no

69

unknown; energy dependent

yes; also in vitro

48,49


no

59,65

or certain N-free media transfer of glutamate grown cells to NH4 + o r media

transfer of derepressed cells to media lacking glucose

Klebsiella, Shigella

(B. A. Hemmings, personal

addition of NH4 + to cells grown under nitrogen

E. coli, Salmonella,

Glutamine

111

lacking N C. utilis.

Glutamate

no

(?)

Agrobacterium

3-dehydrogenase

unknown

inducer

Candida Glucoside

addition of high glucose to derepressed cells

system II a-Glucosidase

93


limitation addition of NH4 to cells

communication) ligand-induced conforma-

no

by dissociation enzymatic adenylylation of

yes; by ph os-

grown under nitrogen

a specific tyrosine residue;

phOTOlytic de-

limitations, or growth

cascade control of adenyl-

adenylylation

on excess N

21,116

tional change, followed 54,120

PRPP

s::

oxidation of enzyme bound

Aerobacter

transfer of induced cells to


amidotransferase Glycerol dehydrogenase

aerogenes

medium containing energy sources and high 02 tension

Z >-l 0 Z 0 '-.:I ttl Z N >

derepressed cells

carboxylase Threonine

-

nine, to the enzyme in the unknown

pressed cells

dehydrogenase

diphosphate

of arginase,

which is induced by argi-

addition of Pi to dere-

IF-3

Ribulose-l ,5-

specific binding

presence of arginine

initiation factor

Pyruvate

unknown

N 2-

B. subtilis

kinase Protein synthesis

addition of NH4 +, gluta-

rubrum, Azoto·

E. coli

addition of glucose, certain keto or amino acids to derepressed cells

binding of pyruvate, followed by dissociation

not in vivo

�

142

SWITZER

tions represent only a part of the various physiological states to which a microbe must be able to respond. So important a regulatory event as inactivation of an enzyme would be much more likely to occur as an organism adapts to a major metabolic transition in which continued activity of a given enzyme becomes un necessary or harmful in a new physiological state.


Shifts in Carbon and Nitrogen Metabolism Perhaps the clearest situation calling for regulation by enzyme inactivation is one in which there is a shift in the nature of the carbon or nitrogen source such that the continued operation of an enzyme involved in utilization of the original metabo lite creates a futile cycle during growth on a new nutrient. An example ofthis occurs following the anaerobic growth of Rhodopseudomonas gelatinosa in the light on citrate (32, 33). During citrate utilization acetate accumulates in the medium; when citrate is exhausted the cells must switch from citrate catabolism to citrate synthesis for the purpose of glutamate synthesis. The first enzyme of citrate catabolism, citrate lyase, has been shown to be inactivated under these conditions, presumably to prevent a futile cycle between citrate synthesis and citrate breakdown. This interpre tation is strengthened by the finding that the inactivating enzyme, S-acetyl citrate lyase deactylase, is specifically inhibited by L-glutamate (34). A similar explanation may be applied to the inactivation of phosphoenolpyruvate carboxykinase (31, 44), fructose 1,6-bisphosphatase (30), and cytoplasmic malate dehydrogenase (19, 22), which is observed when glucose is added to acetate-adapted Saccharomyces cerevi siae cells. Each of these enzymes is involved in gluconeogenesis from acetate, a process that must be blocked to prevent futile cycling in glucose-fermenting yeast cells. Holzer (52) has described this phenomona of glucose-induced inactivation of yeast enzymes in detail and has proposed the term "catabolite inactivation" for it. Another example is the inactivation of the biosynthetic ornithine transcarbamylase in S. cerevisiae by arginase, which is induced when excess arginine is added to cells growing on minimal medium (141). The result is to prevent a futile cycle between arginine synthesis and arginine catabolism. One can imagine many other situations in which an organism is capable of both de novo biosynthesis and catabolism of an essential cellular component that might call for regulatory inactivation to prevent futile cycles; these should provide fertile territory for searching for new inactivation processes. Enzyme inactivation also serves to divert a metabolite at a branch point from one pathway to another. For example, inactivation of isocitrate dehydrogenase in enteric bacteria is provoked by conditions, such as a shift from growth on glucose to growth on acetate, that require utilization of isocitrate in the glyoxalate cycle instead of catabolism through the citric acid cycle (4). Even more common than the situations calling for enzyme inactivation to prevent futile cycles or to t:egulate branch points are those in which a shift in carbon source renders an enzyme unnecessary. In these cases the continued activity of the enzyme may be wasteful to the cell but not directly harmful. Examples are the inactivation of aspartate transcarbamylase ( 1 6, 1 38), aspartokinase (42, 1 2 1), glutamine phos phoribosyl-I-pyrophosphate amidotransferase ( 1 34, 1 35), and threonine dehydra-


1 43

(72) in bacilli when these cells enter stationary phase prior to endospore formation. Such cells appear to be capable of supplying nucleotides and amino acids for spore synthesis from turnover of endogenous RNA and protein. Thus, inactiva tion of enzymes of de novo synthesis presumably spares metabolites and energy. Some members of the group of enzymes subject to catabolite inactivation in yeast (52) and bacteria (20) are enzymes utilizing such secondary catabolites as galactose, maltose, and threonine. These enzymes are inactivated when a preferred catabolite -glucose, fructose, or mannose-is available. The examples of inactivation of enzymes involved in nitrogen metabolism in Table 1 accord well with the generalizations just made. A futile cycle between glutamate synthesis and catabolism in certain yeasts and fungi appears to be prevented by inactivation of NADP- and NAD-dependent glutamate dehydrogenases. NAD dependent glutamate dehydrogenase is induced by growth on glutamate as a nitro gen or nitrogen and carbon source and probably functions in glutamate catabolism (48, 49). If Candida utilis cells are shifted from growth on glutamate to NHt, to other nitrogen sources that lead to NHt, or even to nitrogen starvation, the NAD dependent glutamate dehydrogenase is rapidly inactivated (48, 49). The inactiva tion is stimulated by readily utilizable carbon sources, such as glucose. These observations can be explained by suggesting that the cells are suddenly required to switch from glutamate breakdown to glutamate synthesis under these conditions and that a futile cycle is prevented by inactivation of the dehydrogenase. The reciprocal situation has also been observed; that is, the key enzyme of NHt utiliza tion in yeast and fungi, NADP-dependent glutamate dehydrogenase, is rapidly inactivated in Aspergillus nidulans (59, 65) and C utilis (B. A. Hemmings, personal communication) when cells grown on glucose and NHt are switched to glutamate as a sole carboIJ. and nitrogen source. The absence of glucose is essential for this inactivation, which suggests that futile cycling between glutamate catabolism and glutamate synthesis is most likely to occur in carbon-starved cells. The inactivations of glutamine synthetase in bacteria (54, 120) and yeast (2 1, 1 16), nitrate reductase ( 1 3, 58, 74, 77, 78, 103, 1 1 8, 1 24), and enzymes of nitrogen fixation (66, 89) after the addition ofNHt or otheririch nitrogen sources provide examples of the sparing of metabolic energy by the rapid switching off of reactions that have become unnecessary.


tasc

Differentiation It is likely that the inactivation of enzymes will prove to be associated with microbial differentiation or morphogenesis. Examples from Table 1 are found in bacterial endospore formation (5, 16, 41, 42, 72, 1 2 1 , 1 26, 1 34, 138), endospore germination and outgrowth (93), and myxospore formation (94, 1 05). Protein degradation and inactivation of specific proteins during ascospore formation in yeast have been described by Betz & Weiser (Sa). Examples from differentiating slime molds have been reviewed elsewhere (2, 43, 64, 1 25). In the instances cited the differentiation is generally elicited by a change in the carbon or nitrogen metabolism, however, and the involvement of enzyme inactivation in an event specifically concerned with microbial morphogenesis has not been proven.

144

SWITZER


Why Inactivation? What advantages are conferred in microbial systems by enzyme inactivation? Why is inactivation found in place of or in addition to repression of enzyme synthesis and inhibition of enzymes by metabolites? The answers must be speculative at this time, but the following considerations seem important. In the case of covalent modifica tion-inactivation the modification is catalyzed by inactivating and reactivating en zymes, which are subject to allosteric control and, in at least one case (120), a cascade control involving yet another covalent modification. The advantages of such systems have been discussed by Stadtman & Ginsburg (120) and by Holzer & Duntze (54). Clearly important among these advantages is the increased sensitivity or mUltiplier effect obtained when the regulatory metabolite acts on an inactivating catalyst rather than directly on the enzyme to be inactivated. Furthermore, involve ment of another protein can provide more effector binding sites. If inactivation of the degradative type is found to involve specific proteins, similar advantages would apply to these systems. Simple repression of a harmful or wasteful enzyme is inadequate to block its effects, especially in cells that are not able to synthesize new proteins rapidly, as is often the case during the various metabolic transitions in Table 1. Inhibition of an enzyme by an appropriate metabolite clearly can and does function to switch off enzyme activity in many situations like those described above. During a major metabolic shift, howev,er, a microbe is often starved until it com pletes adaptation to the new growth condition. In such cases degradative inactiva tion can serve not only to block a wasteful reaction but to provide amino acids for the synthesis of new enzymes and catabolites for metabolic energy. Selective degra dation would provide a far more efficient means of doing this than a general random proteolysis. Modification inactivation does not irreversibly commit a cell to a new metabolic situation since the inactivated enzyme can usually be reactivated quickly without new protein synthesis. Inactivation of the degradative type lacks this flexibility, although resynthesis of the inactivated enzyme is possible under appropriate condi tions. It is not yet clear whether the two types of inactivation processes are predict ably distributed in various physiological situations, but the somewhat longer time and protein synthesis required for reversal of degradative inactivation imposes some restrictions on the situations in which it can occur. MECHANISMS OF INACTIVATION

The development of this field is insufficiently advanced to permit a comprehensive survey of the biochemical mechanisms of enzyme inactivation. Only a few mecha nisms, all of the modification type, are adequately understood. Nonetheless, the variety already found suggests that living systems have evolved many different mechanisms for enzyme inactivation.

Physical Mechanisms Included in the physical mechanism group are all mechanisms of the modification type that result from physical changes in the enzyme, generally as the result of tight



145

binding of a molecule or dissociation of the enzyme, in the absence of covalent bond formation or breakage. Inactivation as the result of very tight binding of a low molecular-weight inhibitor occurs in the inactivation of nitrate reductase upon addition of NH;i- to N03-grown algae cells in the presence of light (77; see also 78). In this case CN- is produced in small quantities, which inactivates the enzyme by binding very tightly to it. The tight binding of pyruvate has been implicated in inactivation and dissociation of the biodegradative threonine dehydratase from E. coli (20), but it is not yet known whether the pyruvate is noncovalently or covalently attached to the enzyme. The binding of a macromolecule, arginase, to biosynthetic ornithine transcarbamylase has been shown to be responsible for inactivation of the latter enzyme in yeast (141). Ligand-induced conformational changes, followed by dissociation to inactive or labile subunits, was proposed as the mechanism for NHt -induced inactivation of glutamine synthetase from C utilis (116).

Chemical Mechanisms Inactivation mechanisms that result from chemical modifications without cleavage of peptide bonds are the best understood group of inactivation processes. Glutamine synthetase from enteric bacteria is inactivated by attachment of AMP in phosphodi ester linkage to the phenolic hydroxyl of a specific tyrosyl residue of the protein (120). This modification reaction is catalyzed by an allosteric enzyme system, which is itself subject to regulation by reversible uridylylation of one of the necessary proteins. Because this remarkable system has been thoroughly described elsewhere (54, 120), it is not discussed in detail hereto Inactivation of the citrate lyase from R. gelatinosa by enzymatic cleavage of acetate from a sulfhydryl group on the enzyme has been demonstrated (32-34). The enzyme responsible for this reaction is under appropriate allosteric control (34). A reactivating activity that catalyzes ATP-dependent attachment of an acetyl group has been described but is not yet fully characterized (33). ATP-dependent phosphorylation and inactivation of the pyruvic dehydrogenase complex from Neurospora crassa. as well as enzyme reactivation by a phosphatase, have been reported (142). This system strongly resembles the regula tion of mammalian pyruvic dehydrogenase by a simlar phosphorylation mechanism. Another probable instance of inactivation by phosphorylation has been discovered in studies of the isocitrate dehydrogenase of E. coli (H. C. Reeves, personal commu nication). It is likely that the inactivation of NAD-dependent glutamate dehydroge nase from C utilis involves a reversible covalent modification, but its nature is not yet known (48, 49). Inactivation by chemical changes involving nonprotein prosthetic groups is a possible mechanism in two cases in this review. Mandelstam (82) has proposed that the inactivation of lysine decarboxylase in Bacillus cadaveris results from loss of the pyridoxal phosphate cofactor. Turnbough & Switzer proposed (135) that the oxygen dependent inactivation of glutamine phosphoribosylpyrophosphate aminotrans ferase in B. subtilis is the result of oxidation of tightly bound Fe2+.

Degradative Inactivation: The Role 0/ Proteolysis and Turnover One of the most interesting and least understood of inactivation mechanisms is the degradative type. Although the general phenomenon of protein turnover in mi-


146

SWITZER

crobes has been well studied, there are only a few instances in which evidence has been offered for the disappearance of specific enzyme proteins as part of in vivo inactivation processes. These are the inactivation of aspartate transcarbamylase from B. subtilis (7, 1 26; M. R. Maurizi, J. S. Brabson, R. L. Switzer, unpublished data), isocitrate lyase of Chiarella (62, 130), cytoplasmic malate dehydrogenase of S. cerevisiae (88), NADP-dependent glutamate dehydrogenase of C. utilis (B. A. Hemmings, personal communication), lysine-sensitive aspartokinase of E. coli (127a), protein synthesis initiation factor IF-3 of E. coli (109a), and the assimilatory nitrate reductase of Neurospora (11 8). It is very likely that others of the examples listed in Table I belong in this category and that many undiscovered examples exist. The difficulty in describing mechanisms of this class is that none of them is well understood in biochemical terms. It is not known whether inactivation results directly from a peptide bond cleavage or whether degradation occurs subsequent to another physical or chemical modification, such as those described in the previous paragraphs. The particular proteinases that are involved in vivo have not been identified. The molecular basis of the regulation of the inactivation is not known. It is clear in at least one case (126, 138) that the protein does not tum over continuously. The degradation and turnover of an enzyme must involve many individual steps, but no intermediates have been identified from in vivo studies. Clearly the solution to these problems is one of the most important challenges in this field. Some progress has been made in characterizing the proteolytic enzymes of seme microbes in which inactivation processes occur in vivo (reviewed in 53). Although there is a substantial gap between our knowledge of the action of these proteinases in vitro and our ability to assign roles to them in inactivation or turnover processes in vivo, it is certain that proteinases are involved at some stage in degradative inactivation. The proteolytic system of yeast is perhaps the most thoroughly studied. The various proteinases, their specific protein inhibitors, the subcellular compart mentalization of these proteins, and cultural conditions governing their levels in cells have all been described (53). The participation of two of these proteinases in the inactivation of tryptophan synthetase in vitro has been demonstrated ( 108), but this process appears to proceed rather slowly in vitro (46). Two of the enzymes subject to catabolite inactivation in yeast have been shown to be proteolyzed by yeast proteinases (63, 86, 87), and there is evidence that one of these, malate dehydro genase, is degraded in vivo during inactivation (88). Yet the specificity of the pro teinases in vitro does not account for the selectivity and regulation of the catabo lite inactivation process. Inactivation of enzymes of this group can be induced by glucose in yeast spheroplasts (84), but no such regulation can be demonstrated in lysed spheroplasts (H. Matern and H. Holzer, personal communication). The possi bility that the selectivity of catabolite inactivation in yeast is actually imposed at the level of transport �nto the vacuole, where the proteinases are concentrated, has been put forward by Holzer (52). This mechanism cannot account for the selectivity of the degradative inactivation of aspartate transcarbamylase in B. subtilis, however, because no such subcellular compartments exist in this organism. The proteinases of B. subtilis have been characterized by biochemical and genetic techniques (17, 45, 98, 1 02, 127). There is at present, however, no evidence linking


147

these proteinases to an inactivation process. Evidence obtained with proteinase deficient mutant strains argues against their involvement in the primary step of inactivation of aspartate transcarbamylase or glutamine phosphoribosyl- l -pyro phosphate amidotransferase (16, 134, 138).


Unknown Mechanisms Although it is likely that most inactivation mechanisms will be found to fit into one or a combination of the above categories, the discovery of other mechanistic types or major new mechanistic features must not be excluded. At least six of the examples in Table 1 present unknown biochemical mechanisms in which the inactivation process requires metabolic energy. This requirement generally has been demon strated with the use of inhibitors. Its meaning in biochemical terms is obscure. Such a requirement could represent (i) a direct involvement of ATP or reducing equiva lents in a covalent modification, (ii) a requirement for energy for protein or nucleic acid synthesis, which has been generally excluded by use of inhibitors, (iii) a require ment for energy to maintain a pool of regulatory or "signaling" metabolites, or (iv) a requirement for energy for transport of the protein into a subcellular compart ment. Particularly intriguing in this regard is the report of Thurston et al.(130) that the inactivation of isocitrate lyase in Chlorella is not inhibited by 2,4-dinitrophenol, but the disappearance of the protein is. A portion of protein turnover in microbial and mammalian systems is known to be energy dependent (36, 37, SO, 95, 1 10, 122). Generally, it has not been possible to demonstrate this energy dependence in cell free systems (9, 36-38, 56, 95, 96). Curiously, failure to demonstrate inactivation in vitro has also been reported in several of the energy-dependent systems (52, 93, 1 38). Although these observations may be coincidental, there is a possibility that the phenomena of energy dependence of inactivation, energy dependence of protein turnover, and the difficulty of reconstructing both in vitro are mechanistically linked. If so, these systems offer experimental objects for the study of an important area of regulatory biochemistry. METHODS FOR STUDYING INACTIVAnON PROCESSES

General Characterization The characterization of a suspected enzyme inactivation process must include exper iments to establish that a true inactivation takes place. (i) It must be demonstrated that a loss in total activity occurs, i.e. that the specific activity of the enzyme decreases faster than can be accounted for by cessation of synthesis and dilution by the increase of cellular protein. (ii) Extreme care must be taken to demonstrate the stability of the enzyme in question during cell extraction and assay or at the very least to demonstrate that there is no change in the stability of the enzyme in extracts of cells harvested at various times during the apparent inactivation. This is impor tant because the content of stabilizing or-destabilizing metabolites or of degradative enzymes often changes during metabolic transitions of the kinds that induce enzyme inactivation. An example of the extreme care that may be required to avoid artifacts in the assay of an unstable enzyme that is stabilized by metabolites and increases in protein concentration is given in the studies of pyruvate kinase from Bacillus


148

SWITZER

licheniformis (133), an enzyme that earlier had been reported to be inactivated in the stationary phase of growth (5). The appearance of proteinase activities in starved cells is a particularly troublesome problem. Recently, we were able to show that the apparent inactivation of IMP dehydrogenase in sporulating B. subtilis cells is an artifact caused by proteinase activity during preparation of cell extracts (85). Some methods of detecting and avoiding such artifacts were presented. (iii) Because the intracellular concentration of some enzymes appears to be regulated by excretion in the differentiating slime molds (43, 64, 91), one must determine whether an enzyme that disappears from microbial cells can be found in the growth medium and, when findings are negative, demonstrate that the enzyme in question could have been detected at the expected levels in the culture medium by the assay used. At least one instance of inactivation of an extracellular enzyme by an extracellular protein, possibly a proteinase, is known (28). (iv) Inactivation of an enzyme can be mimicked by transfer of the enzyme to a subcellular compartment that is impermea ble to assay reagents or discarded during preparation of extracts. A fraction of the soluble glycogen synt�etase of Dictyostelium discoideum undergoes such an associa tion with insoluble cell wall material during culmination (43, 64). Therefore it is necessary to examine unfractionated extracts, cell debris, and extracts prepared by a variety of rupture methods to ensure detection of any enzyme rendered cryptic by transfer to a subcellular compartment. (v) In some cases of modification-inactiva tion systems the altered enzyme shows major changes in cation specificity, pH optimum, Michaelis constants for substrates, and sensitivity to metabolic effectors (48, 54, 120). Hence, it is necessary to determine these kinetic parameters on enzyme samples taken before, during, and near the end of a suspected inactivation process. Except in the case of selective inactivation of isozymes, the finding of such shifts in kinetic properties provides tentative evidence for an inactivation of the modifica tion type (or possibly a modification that is not true inactivation) and argues against degradative inactivation. (vi) A major change in the activity of an enzyme in crude extracts could result from loss of an activator or accumulation of an inhibitor as the cells undergo a metabolic transition. For this reason it is desirable to determine whether dialysis or gel filtration has effects on the activity of extracts of cells harvested before, during, and after a suspected inactivation. The definition I have applied to enzyme inactivation excludes inhibition or loss of activation by dialyzable metabolites, but such phenomena are obviously of interest in the overall regulation of a given enzyme. Experiments in which extracts of cells harvested before the inactivation are mixed with extracts from cells harvested during or after the inac tivation also aid in identifying possible activators, inhibitors, or inactivating en zymes that may be responsible for an apparent inactivation process. In this case effectors of any molecular size will be detected if they are present in excess of the enzyme or act catalytically. The cases of most direct interest are those in which an inactivating protein is found. In view of the possible artifacts in such experiments it is essential to develop criteria for establishing that any inactivation in vitro is the same as that which occurs in vivo. The general characterization of an inactivation process should also include experi ments that define the range of conditions in which inactivation occurs, the specificity


149

of nutritional effectors, and the situations in which the enzyme reappears. Such studies often define a probable physiological function for the inactivation and may provide clues as to the metabolites that are required for the inactivation to occur.


Effects of Inhibitors Further analysis of an inactivation process is usually aided by use of metabolic inhibitors. The general principle is to determine the biochemical requirements of an inactivation process by adding inhibitors of specific metabolic sequences prior to the inactivation, while the inactivation is in progress, and prior to a manipulation that leads to reappearance of the activity, if one can be found. For studies of a require ment for RNA and protein synthesis, rifampin and chloramphenicol, respectively, have found extensive application with bacterial systems, actinomycin D and cy cloheximide with eukaryotic systems. A requirement for metabolic energy has been revealed by use of anaerobiosis, uncouplers, and inhibitors of electron transport (e.g. 30, 48, 68, 93, 94, 119, 130, 138). Such inhibitors are most useful if they are used under conditions in which the cells are totally dependent on oxidative metabolism for energy generation, i.e. when fermentative pathways cannot function. The use of inhibitors of glycolysis has proven less satisfactory in experiments with whole cells. The proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF) has been used to test the involvement of proteolysis in inactivation in vivo (10, 94), but in view of a report that PMSF has pronounced effects on energy metabolism in bacteria (114) caution in interpretation of results is required. This last observation points out a serious problem in the use of metabolic inhibitors, namely, few of them are fully specific in their action. Cycloheximide, in particular, has been shown to affect overall metabolism in yeast cells (22). Rifampin may have unknown side effects since this antibiotic has been shown to kill B. subtilis cells rather than act as a bacterio static agent (12). Hence, it is desirable to document the effectiveness and specificity of action of a given inhibitor whenever possible. A range of concentrations should be examined. In addition, it is advisable to test a variety of inhibitors and to supplement inhibitor studies with the use of appropriate mutant strains.

Use of Mutant Strains Although mutant strains have not been extensively used in the characterization of inactivation processes, their use is one of the greatest potential advantages in study ing inactivation in microbes. Mutant strains with a defect in a given metabolic sequence can be used in physiological studies to define the requirements for inactiva tion or reappearance of an enzyme in much the same way as specific inhibitors are used but without many of the disadvantages of inhibitors. For example, mutant strains defective in enzymes of the tricarboxylic acid cycle were used to define the requirement for energy metabolism in the inactivation of aspartate transcarbamylase in B. subtilis (138). Duntze et al (19) used a yeast tryptophan auxotroph to show that protein synthesis was not required for catabolite inactivation of malate dehy drogenase but was required for reappearance of enzyme activity. Mutant strains can be used to test for involvement of metabolites in situations where no specific inhibi tors are known. Rel- mutant strains have been used to demonstrate that an inactiva-


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tion does not require a functional stringent response (138), for example, a conclusion that would be difficult to reach in other ways. Mutant strains deficient in specific intracellular proteinases (45, 127) have also proven useful in probing possible in volvement of these proteinases in inactivation of particular enzymes (16, 134, 138). This use of proteinase-deficient mutants has several uncertainties: the possibility of pleiotropic effects in the mutant strains; the fact that most strains are not totally lacking in the proteinase activity; and the overlapping specificity of multiple protei nases in the species examined. With the exception of the detailed genetic studies of the inactivation of glutamine synthetase by Magasanik and his colleagues (15,25,26,61,80, WI, 123),very few attempts to isolate mutant strains defective in inactivation or reappearance processes have been reported. The results of the genetic studies with glutamine synthetase in Klebsiella aerogenes point the way to a general and powerful method for analyzing enzyme inactivation systems, at least those involving specific inactivating enzymes. In the case of glutamine synthetase, glutamine auxotrophy will result not only from mutations in the structural gene for glutamine synthetase (ginA) but also from defects in the enzymes that reactivate the enzyme by catalyzing removal of the adenylyl group (glnB, glnD). Another class of mutations (glnE) was isolated from revertants of the glnB class; these were showri to be defective in the adenylylating enzyme. The effectiveness of the use of glutamine auxotrophy as a selective pheno type was reinforced in this case by the fact that adenylylated glutamine synthetase is not only inactive but serves to repress synthesis of glutamine synthetase. The discovery of roles for adenylylated and deadenylylated forms of glutamine synthe tase in regulation of transcription of certain genes coding for enzymes involved in nitrogen metabolism was an important additional contribution from these studies

(79). The application of the approach developed by Magasanik and colleagues in the study of glutamine synthetase to other inactivation systems that involve reversible modification inactivation should be straightforward and fruitful as long as mutant strains that are defective in the activity of the enzyme that is inactivated can be selected easily. In the case of enzymes subject to degradative inactivation, there is no precedent to guide our thinking. Since in such cases reappearance·of the enzyme results from new synthesis, defects in reactivating enzymes cannot be expected. R ather one would expect mutations in the structural gene of the target enzyme rendering it hypersensitive to the inactivating system, and mutations in the inac tivating system so that it has lost its normal selectivity and destroys the target enzyme at inappropriate times. Genetic mapping and analysis of the properties of appropriate diploid strains should be valuable adjuncts to biochemical characteriza tion of such mutant strains. An interesting alternative approach was taken by van de Poll et al (136), who isolated a mutant strain of S. cerevisiae that was unable to grow on glucose but was able to grow on glycerol or ethanol. This strain is incapable of carrying out the inactivation of fructose 1,6-bisphosphatase that normally follows addition of glucose to derepressed cells (30, 86). Catabolite inactivation of maltose permease (104) is normal in the mutant cells,but the inactivation of cytoplasmic malate dehydrogen-



151

ase and phosphoenolpyruvate carboxykinase is also blocked (31). The mutant cells are capable of normal glucose uptake, and no defects in the enzymes of glycolysis or gluconeogenesis have been detected (K. W. van de Poll, personal communica tion). The intracellular levels of ATP drop sharply in the mutant strain (unlike the wild type) when glucose is added to glycerol-grown cells. This result suggests that failure to carry out the normal catabolite inactivation results in an ATP-splitting futile cycle between glycolysis and gluconeogenesis. The nature of the biochemical defect in this interesting mutant strain remains to be defined; its identification might yield valuable clues to the nature or regulation of the catabolite inactivation process in yeast. The general approach to selecting mutant strains defective in inactivation processes adopted by van de Poll et ai, namely, selecting for the nutritional limita tions imposed by failure to inactivate an enzyme under given conditions, should be applicable to other situations.

Reactivation In Vivo and In Vitro Culture conditions generally can be found under which the activity of an inactivated enzyme returns to the cells. If one can determine whether this reaction requires de novo synthesis of the enzyme, one can distinguish between inactivation processes of the modification type and of the degradative type. Most investigators have ap proached this problem by exposing cells to reactivating conditions in the presence of antibiotic inhibitors of RNA or protein synthesis. Restoration of activity in the presence of inhibitors implies reactivation of already existing, inactive enzyme mole cules. It is necessary to document the effectiveness of the inhibitory conditions used. The converse result, failure to observe restoration of activity, may indicate that the inactivation is of the degradative type and that restoration requires de novo synthe sis. This is a crude criterion, however, because protein synthesis could also be required for formation of a reactivating enzyme and because the inhibitors may have secondary effects on metabolism. Reactivation of an inactivated enzyme in cell extracts, incubated under conditions where cell-free protein synthesis is excluded, provides strong evidence that an inactivation is of the modification type and is often a valuable technique for learning the biochemical nature of the inactivation process. For example, the activity of adenylylated glutamine synthetase can be restored by treatment of the enzyme with phosphodiesterase (120). Reactivation of citrate lyase was accomplished chemically by treating the inactivated enzyme with acetic anhydride (32). The inactivation of the NAD-dependent glutamate dehydrogenase from C. uti/is appears to result from a covalent modification, although the nature of this modification has not yet been defined (48, 49). The inactivated enzyme can be readily reactivated in vitro; a reactivating protein has been separated from the dehydrogenase (48, 49).

Following the Fate of the Enzyme Protein During Inactivation Biochemical analysis of an enzyme inactivation occurring in vivo or in crude cell extracts suffers from the difficulty that the experimenter has only one tool for following the inactivation, namely, assay of catalytic activity. More can be learned about the biochemical nature of an inactivation and events subsequent to it if one


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has a specific means of following the fate of the enzyme protein independently of its loss of catalytic activity. Is the protein accumulated in an inactive form or is it degraded? If an inactive protein is accumulated, what physical and/or chemical changes has it undergone? Several approaches to this problem have been used, the most general of which is immunochemical. It is unlikely that loss of catalytic activity in a modification inactivation will result in loss of all the antigenic determinants of the enzyme protein. For example, adenylylated glutamine synthetase is immuno chemically identical to the unmodified form (132). Hence, antibodies specifically directed against the native enzyme can be used in such cases to identify the accumu lation of inactive cross-reactive material. On the other hand, if the enzyme is extensively degraded during or after inactivation, it will not be detected by antibod ies directed against the native enzyme. There are some uncertainties in this last conclusion. One does not know a priori how extensive the denaturation or degrada tion of an enzyme must be for it to escape detection by immunochemical techniques. Loss of native quaternary and tertiary structure is often associated with a marked decrease in antigenicity of globular proteins, although peptide fragments of enzymes retain the ability to react with antibodies in many cases (14). Thus, detection of modified, denatured, or degraded products of enzyme inactivation processes should be possible in some cases, but failure to detect such products does not permit one to conclude that degradation and turnover of the enzyme has occurred. An ap proach deserving further study is the use of denatured enzyme as the antigen as a possible means of obtaining antibody directed against sequential determinants rather than conformational determinants (14). The methods used for detection and quantitation of antigen-antibody complex formation can lead to contradictory con clusions, so it is desirable to use more than one method. In our own research on the inactivation of aspartate transcarbamylase in B. subtilis (7, 126; M. R. Maurizi & R. L. Switzer, unpublished experiments), we have assayed immunoprecipitation by Coomassie Blue staining and densitometry of immunoprecipitates after e1ector phoresis on sodium dodecyl sulfate-polyacrylamide gels and by counting gel slices after electrophoresis of immunoprecipitates from cells labeled with radioactive amino acids. These methods were useful because the aspartate transcarbamylase subunit is well resolved from the immunoglobulin chains and because fragments derived from dragradation of the enzyme that were precipitable by the antibody would have been detected. The results showed clearly that immunoprecipitable protein disappears from the cells in parallel with catalytic activity. A more com monly used immunochemical technique is to use the ability ofthe antibody to inhibit the enzyme to be assayed as a measure of the antigen present in an extract (72, 88, 118). This approach assumes that an inactive, cross-reactive protein will also adsorb antibody, so that an accumulation of inactive material will result in an increase in the quantity of antibody required to inhibit a unit of catalytic activity. In our experiments with aspartate transcarbamylase, this method also indicated that inac tive, cross-reactive material did not accumulate. A potential difficulty with this approach is the possibility that the technique measures the effects of only that subclass of antibody molecules that bind at or near the active site. Since the active site is probably altered during inactivation, failure of this subclass to interact with



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the active site could possibly lead one to yonclude incorrectly that all antigenic sites had disappeared. Other techniques such as microcomplement fixation (73, 140) or indirect immunoprecipitation ( 1 1 3) have promise as methods for following the fate of inactivated proteins, because these methods can detect soluble antigen-antibody complexes. Another technique of potentially general usefulness for determining whether degradation and synthesis occur simultaneously is the density shift technique used by Williams & Neidhardt (144) to demonstrate the turnover of aminoacyl tRNA synthetases in E. coli cells. The cells were grown on 80% deuterium oxide until the proteins were uniformly labeled with deuterium, then they were transferred to normal medium under the conditions of interest. Enzyme synthesized before the shift (density 1 .33) can be separated from newly synthesized enzyme (density 1 . 30) by centrifugation on isopycnic CsCI gradients and quantitated by enzymatic assay. As described, the method would not determine whether an inactivated protein accumulates in the cell, but a simple variation could be used to learn whether an inactive, density-labeled enzyme regains activity during transfer to normal medium under reactivating conditions. Unfortunately, Brown et al (10) were unable to use this technique in studies with yeast cells because of failure of the cells to grow in deuterium oxide medium. Other approaches to following the fate of enzyme protein during an inactivation process are less general. In favorable ca!;es, one may be able to separate the protein in question from all other proteins in the cell by electrophoresis or combined electrophoretic and isoelectric focusing techniques. As methods for reproducible separation of complex protein mixtures are more highly developed (e.g. I, 92) this approach may become quite useful. I am aware of only two instances in which electrophoretic isolation of an enzyme protein was used to characterize an inactiva tion process. When Chlorella cells are adapted to growth on acetate as a sole carbon source, isocitrate lyase is produced to a level of about 7% of the total cell protein. This protein is readily identified as a discrete band after polyacrylamide gel electro phoresis of cell extracts, so that the fate of the enzyme protein can be followed independently of enzyme activity (62, 1 30). This technique has been used to show that the enzyme is normally degraded during or subsequent to glucose-induced inactivation of the enzyme, but that 2,4-dinittophenol apparently protects the inac tivated enzyme from degradation (1 30). An electrophoretic separation was also used by Orlowski & Goldman to determine the fate of 35S-labeled glucose-6-phosphate dehydrogenase during inactivation that occurs during germination of B. cereus spores (93). They concluded that the enzyme was not degraded during inactivation. This conclusion is dependent on the assumption that the enzyme band, which was located on polyacrylamide gels by an activity stain, was free of other cellular proteins. This assumption was not documented in the paper, however. =

=

Reconstruction In Vitro A satisfactory understanding of any enzyme inactivation process cannot be achieved until it has been reconstructed in vitro from highly purified components in such a fashion that its physiological operation and regulation can be reproduced by appro-

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priate manipulation. This goal is nearly reached in the modification inactivation of glutamine synthetase (112, 120). Progress toward this goal has been made in several ' other cases, all of the modification-inactivation type. For those systems involving degradative inactivation the current status is much less satisfactory. Problems in reconstruction of catabolite inactivation in yeast have been discussed above, as have those dealing with energy-dependent systems. In all cases it is necessary to demon strate that the reconstructed process faithfully reproduces the inactivation that occurs in vivo. This means that biochemical equivalents for the physiological re quirements and regulation of the inactivation must be shown for the in vitro process. CONCLUDING COMMENTS

It has been the purpose of this review to show that the selective inactivation of enzymes is a general and important regulatory mechanism in microbes and to review what is known concerning the mechanisms of inactivation. Unlike other major types of regulation, inactivation processes do not appear to possess an appealing mecha nistic unity. Perhaps for this reason the importance of this class of regulatory processes has been slow in achieving recognition. The systems involving reversible covalent modification of enzymes, all of which were unknown a decade ago, are the best studied and have greatly expanded our understanding of metabolic regulation in microbes. The magnitude of the biochemical challenge lying ahead for this field can be readily appreciated by simply noting the number of inactivation processes of unknown mechanisms listed in Table 1 . Particularly challenging are those mecha nisms that appear to involve the degradation of enzymes and the participation of metabolic energy. The elucidation of the molecular details of these processes particularly the basis of the selectivity of degradation---could very well provide insight into the problem of protein turnover in all living systems. ACKNOWLEDGMENTS A major

portion of this review was prepared while I was a Fellow of the John Simon Guggenheim Memorial Foundation and a guest in the laboratory of Helmut Holzer, Biochemisches Institut, University of Freiburg, Germany. I am grateful to Prof. Holzer and his colleagues for their hospitality and many stimulating discussions of the subjects reviewed. My own research in this area has been supported by grant AI 11121 from the National Institutes of Health.


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