Zinc proteins: enzymes, storage proteins, transcription factors, and replication proteins.

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1992

Annu. Rev. Biochem. Copyright ©

1992. 61:897-946

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Annu. Rev. Biochem. 1992.61:897-946. Downloaded from www.annualreviews.org by University of Melbourne on 03/13/13. For personal use only.

ZINC PROTEINS: Enzymes, Storage Proteins, Transcription Factors, and Replication Proteins Joseph E. Coleman* Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510 KEY WORDS:

mechanism of action of zinc enzymes, zinc fingers, hormone receptors, metallothionein, DNA- binding protein�

CONTENTS INTRODUCTION .....................................................................................

ZINC ENZyMES ............................. . . ............................... ......... . ...... . ...... Summary . ..... .................... .......... ........................................ ............ .... Catalytic Functions of Zinc-Zinc Coordinated Water as a Nucleophile . . . . . . . .. . .... . .

Multi-Zinc Sites in Enzymes.. . . . . . ... . . . . . . . . ... . . . . . .. .. .... . . .. .. . . . . . . ... .. . . . . ... . .. . . . ... .

898 898 898 900 908

ZINC STORAGE AND TRANSPORT: ROLE OF METALLOTHIONEIN...............

920

ZINC-REQUIRING TRANSCRIPTION FACTORS............................................

923 923

Classes of Zinc-Containing Transcription Factors .. . . .................. . . . ............ .... Solution Structures of the Zinc-Containing DNA-Binding Domains of Transcription Factors as Determined by 2D NMR Methods................ Crystal Structure of the Three Zinc Fingers of the Zif 268-DNA Complex . . .. . . . . . . . Crystal Structure of the DNA-Binding Domain of the Glucocorticoid Receptor Complexed with the DNA of the Glucocorticoid-Responsive Element (GRE) . . . . .. . . . . . . .. . . . .. . ........ ..... ........................... ...........

924 932

935

ZINC PROTEINS BINDING TO SINGLE-STRANDED NUCLEIC ACIDS . . . . . . . . .. . . . Gene 32 Protein from Bacteriophage T4 .. . . ................. .... .. . . . ............. . ........

938

Retroviral Nucleocapsid Proteins . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

938 939

SUMMARy ............................................................................................

940

"Original work in the author's laboratory was supported by NIH grants DK09070 and GM21919.

0066-4154/92/0701-0897$02.00

897

898

COLEMAN


INTRODUCTION

Since 1869 zinc has been known to be an esssential trace element for eukaryotes (1). The presence of zinc in an enzyme was recognized for the first time in 1940 by Keilin & Mann, who discovered zinc in carbonic anhydrase (2). This remained the sole example of zinc functioning in a biologically important macromolecule until Vallee and coworkers described several other zinc metalloenzymes in the 1950s; these included carboxypeptidase A (3), alcohol dehydrogenase (4, 5), and alkaline phosphatase (6, 7). There are now more than 300 known zinc enzymes (8). More recently zinc has been dis covered to play structural and functional roles in an entirely new class of protein molecules, namely a variety of eukaryotic transcription factors. Sever al proteins involved in DNA replication and reverse transcription also have been discovered to be zinc proteins. This large increase in the number of proteins known to contain zinc with functions extending from catalysis of metabolic pathways and macromolecular synthesis to the regulation of gene expression bring the known functions of zinc more into line with the fact that the average adult human body contains 2.3 g of zinc compared with 4 g of iron. Thus zinc is the second most abundant trace metal in most higher animals, and its functions are beginning to catch up with its abundance. ZINC ENZYMES Summary

The earliest zinc enzymes to be discovered contained a single zinc atom at the active site, e.g. carbonic anhydrase and carboxypeptidase A. The ligands to the zinc ions in these two proteins consist of three H nitrogen atoms in carbonic anhydrase (9) and two H nitrogen atoms plus both oxygens of the ,,-carboxylate of a glutamate residue in carboxypeptidase ( 10, 1 1). The coordination sphere is completed in both enzymes by a water molecule to form a tetrahedral complex in the first instance and a five-coordinate complex in the other. A large majority of the zinc enzymes discovered follow this pattern, in that they have single zinc sites consisting of a combination of N and 0 ligands with a solvent water molecule completing the coordination sphere. The role of zinc in the structure and function of enzyme molecules is not limited to a single site. Alcohol dehydrogenase contains two zinc ions per subunit, only one of which is present at the active center ( 12, 1 3 ) . The other zinc site is located ca. 14 A away from the active center and has been termed a "structural site." The zinc at the latter site is tetrahedrally coordinated to the -S - donors from four Cys residues and does not have a coordinated solvent water. The active-center zinc, although following the typical pattern of three protein ligands and a solvent water molecule, is ligated by the -S- donors


ZINC PROTEINS

899

from two Cys residues and the N-3 of a His residue ( 1 3). Thus sulfur coordination, expected for lIB metal ions , does occur in zinc enzymes , but not as frequently as might have been expected. The great majority of catalytic zinc sites are mixtures of nitrogen and oxygen ligands. Asparate transcarbamylase is an enzyme that contains only a structural zinc ion. This zinc, one in each of the six regulatory subunits , is coordinated by the -S- donors from four Cys residues and stabilizes the conformation of the peptide loops which form the interface between the regulatory (R) and catalytic (C) subunits (14). The R and C subunits dissociate when Zn is removed. The constellation of amino acid side chains participating in zinc coordination at enzyme active sites has been reviewed by Vallee and Auld (8). The ligands and their spacing in the polypeptide chains have been used to provide a significant guide to the molecular topology of these enzyme mole cules. This brief introduction to the zinc enzymes that contain isolated zinc

sites serves to put in perspective the discovery in the last several years of zinc

enzymes that contain three closely spaced zinc ions , two of which form a binuclear pair via a bridging carboxylate ligand. There are now available crystal structures of three zinc enzymes, all involved in various aspects of phosphate ester transformation, in which these sites have been found: alkaline phosphatase ( 15) , PI nuclease ( 16), and phospholipase C (17). These multi zinc sites add a new dimension to the participation of zinc in complex reaction mechanisms. Before leaving this brief overview of zinc proteins, it should be recalled that very solid data now exist showing that the bacterial multisubunit RNA polymerases, as well as the eukaryotic RNA polmerases II and III, contain two zinc ions associated with their largest subunits, f3 and f3' , following the nomenclature applied to the bacterial enzymes (18-20). The two largest subunits of the eukaryotic enzymes show extensive homology to f3 and f3' of the Escherichia coli enzyme. Thus zinc assumes a central role in transcription. One of the zinc sites is almost certainly located in the f3' subunit and is a tetrahedral sulfur-containing site based on the presence of d-d absorption bands for the Co(ll) derivative typical of a tetrahedral complex along with -s- � Co charge transfer bands (20). A cluster of C residues containing two -C-Xr C- sequences located near the N terminus of the f3' polypeptide and preserved in the largest subunit of the eukaryotic polymerases is likely to contribute the ligands for this site (21). The nature of the second zinc site is less clear. The ready displacement of this zinc by mercurial groups suggests the presence of sulfur coordination; however, the site does not appear to be tetrahedral since no typical tetrahedral d-d absorption spectrum is observed in the Co(II) derivative (21). The zinc at the tetrahedral sulfur site in the E. coli enzyme cannot be removed without

RNA POLYMERASES


900

COLEMAN

denaturation of the protein. In contrast, the second zinc is easily removed by treatment with mercurial compounds followed by dialysis against thiols to remove the mercurial compound. Removal of this zinc has no effect on the standard transcription assays carried out in the test tube (2 1 ). There are simple one-subunit RNA polymerases which clearly do not contain zinc. The enzyme from bacteriophage T7 shows no zinc dependence (22), and the enzymes from T3 and SP6 are probably similar. This finding suggests , but does not prove , that the zinc in the multisubunit RNA polymerases may not participate directly in the polymerization reaction, but may be required for the folding of certain subdomains of the enzyme or for their interactions with other subunits or proteins of the transcription complex. Exactly such functions are now known to characterize the role of zinc in a variety of eukaryotic transcription factors, as outlined later in this review. Catalytic Functions of Zinc-Zinc Coordinated Water as a Nucleophile

When carbonic anhydrase was the only zinc en zyme known, several proposals were put forward that the function of the Zn ion could be to generate a Zn hydroxide at neutral pH which would attack the adjacent CO2 molecule. This was an attractive mechanism, since the attack of -OH on CO2 proceeds rapidly at pH> 10 (k 8 . 5 x 1 03 S-I M- 1 ). Variants of this mechanism were suggested in 1 949 by Smith (23), in 1959 by Davis (24), and in 1961 by DeVoe and Kistiakowsky (25). Following these early suggestions, it was thought more likely that zinc enzymes used amino acid side chains in concerted reactions in which the Zn was acting as a Lewis acid directly coordinating a group on the substrate and withdrawing electrons, thus labilizing a bond. While at least one open coordination site on the enzyme-bound zinc would be required for such a function, the solvent water, expected to be present in the absence of the substrate, was thought likely to be displaced by substrate. Thus an amino acid side chain, probably the imidazole ring of H rather than a Zn-coordinated H20 molecule. was favored as being responsible for the sigmoid pH activity profile of carbonic anhydrase (apparent pKa of 6 . 5 to 8 depending on conditions) (25-27) . Two general mechanisms for carbonic anhydrase can be formulated: either the attack of H20 on CO2 to produce the netural carbonic acid or the attack of -OH on CO2 to produce the bicarbonate anion. Carbonic acid appears highly unlikely as a substrate in the reverse reaction because its concentration is so low in the pH range in which the enzyme is active that protonation of the predominant species, HC03 - , would not supply the substrate fast enough (27, 28). The zinc ion at the active center of carbonic anhydrase is tetrahedral ly coordinated at the bottom of a 1 2-A-deep cavity as shown in Figure 1 , taken from the 2.0-A crystal structure of human carbonic anhydrase II (29 ,

CARBONIC ANHYDRASE

=


ZINC

PROTEINS

901

30) . The amino acid side chains of closest approach to the zinc ion are H64, Y7, El06, and T 1 99. The cavity has a hydrophobic and a hydrophilic side , the former centered around L198 and the latter around H64. Extensive studies have ruled out the pKa of H64, as well as that of other amino acid side chains , as being responsible for the apparent pKa of the activity (for complete coverage, see reference 3 1) . It is now generally accepted that the pKa reflected in the activity profile is that of a Zn-coordinated water molecule, Zn-OH2 :;;::= Zn--OH + H +. Thus the carbonic anhydrase mechanism can be written as in Eq. 1. -H+

Zn-DH2

� Zn- -OH + CO2 � Zn-HC03 - + H20 � Zn-DH2 + HC03 +H+

Proton transfer

1.

Ligand exchange

Several metal-centered phenomena suggested early that the Zn-OH2 :;;::= Zn--OH + H+ eqUilibrium accounted for the EH � E + H+ transition of carbonic anhydrase. When the anions CN- and -SH or their respective acids,

Figure 1

Structure at the active center of carbonic anhydrase (29).

902

COLEMAN

HCN and H2S, bind to the active-site Zn ion, protons are taken up or released, respectively

(32). Fitting of the resulting biphasic release or uptake or protons

as a function of pH requires a combination of the pKas of HCN and H2S

(9 . 3

and

6.9 , respectively) and a single pKa on the enzyme. The enzyme pKa is the same for both agents, 7.5 to 8 depending on the isozyme. A protonatable

group whose pH equilibrium is directly coupled to formation of a complex between the active-center metal ion and a monodentate anion can best be


explained in terms of the proton dissociation from a coordinated water molecule

(32) .

Second, carbonic anhydrase displays esterase activity against paranitro phenyl acetate with a pH-activity curve similar to that for CO2 hydration

(33).

A substitution of Cd for Zn in the enzyme shifts the apparent pKa of activity from pH ca.

7.5 to ca. 9.3 (34). An alkaline shift in a proton equilibrium of

this magnitude induced by a metal ion substitution seems only satisfactorily explained by the Zn-OH2 � Zn- -OH + H+ hypothesis. The much softer

metal, Cd, would be expected to be much less effective in lowering the pKa of a coordinated solvent. If the proton acceptor were bulk water in the proton transfer step, the

reaction, dependent on a group with a pKa of 6.5 to 7.5, should not proceed -1 faster than ca. 103 S (28). In fact, hydration reactions as fast as 105 S-1 have been observed for the isozyme I and 106 s -1 for isozyme II of human carbonic anhydrase. These high rates have been explained by the fact that proton transfers during initial rate measurements are transfers to buffer with a pKa near neutral pH, rather than to the H20. The lattcr involves the H30+ species with a pKa of ca. -1. 7, thus limiting the transfer rate to ca. 103 s -1. Using 13C and 180 isotope exchange methods, Tu and Silverman (35, 36) have measured carbonic anhydrase rate constants at equilibrium and have shown that as buffer is removed,

transfer limit of 103 to

104

kcat falls toward the theoretical proton

S-I. In the presence of buffer, some step in ligand

exchange may become rate limiting. Ligand exchanges such as that of HC031) most probably proceed through expansion of the coordination

for H20 (Eq.

sphere. Recent high-resolution crystal structures of the bicarbonate complexes of both Zn and Co carbonic anhydrase have shown that bicarbonate is a monodentate ligand (A. Liljas, personal communication). The Zn enzyme bicarbonate complex is tetrahedral, whereas the Co enzyme-bicarbonate complex is five coordinate with a solvent water occupying the fifth position. Thus a five-coordinate intermediate is possible and Co has shifted the equilib rium in favor of a five-coordinate species. The crystal structures of carbonic anhydrase show that the active-center cavity of carbonic anhydrase contains organized water molecules that form a network of hydrogen bonds linking the side chains of

H64, Y6, E 1 06, and

T l99 and the Zn- -OH (Figure 1). Several effects of this chain of hydrogen


ZINC PROTEINS

903

bonds on the reaction mechanism have been postulated (37-39) . (a) The network could serve to orient the nucleophilic hydroxide so that it is properly positioned relative to the carbon of the linear CO2 molecule in the bottom of the cavity. (b) The network may provide a pathway for proton transfer to solvent or buffer via H64. Mutants of H64 show a decreased kcat that is adequately explained by a less facile proton transfer step; e.g. the mutation 4 H64A in human carbonic anhydrase II (HCAII) reduces the kcat to ca. 1 0 S- I . (c) The precise hydrogen bond stereochemistry as well as the length of hydrogen bonds in a water network connected to the Zn- -OH or Zn-OH2 can affect the rate of proton transfer from the Zn-bound water and also its pKa, e.g. by stabilizing Zn-OH2 over Zn--OH (39). The discovery of carbonic anhydrase III (CAllI) in skeletal muscle, representing as much as 20% of cytosolic protein, has given important insights into the features around the Zn-OH2 at the active center of carbonic anhydrase that influence the rate of reaction (40, 4 1 ). Although similar in structure to other isozymes of carbonic anhydrase, the CO2 hydration activity of CAllI is 500-fold lower (2 x 103 S- I) than that of isozyme II. Various physicochemical parameters including anion binding and d-d absorption spec tra of the Co(lI) derivative of isozyme III show that the enzyme remains in the alkaline (E) or active form to pH values as low as 5 . 5 (42). Yet all evidence still suggests that the E form of the enzyme is a Zn--OH species. The crystal structure of carbonic anhydrase III has now been solved, and it is indeed very similar in general structure to that of isozyme II (43). The two polypeptide chains have 58% amino acid identity, and the root mean square (rms) devia tion of all main-chain atoms in the two enzymes is 0.92 A. Three striking amino acid substitutions were found in the three residues near the zinc ion, H64 � K64, N67 � R67, and L198 � F I 98 . Two of these residues , K64 and R67, are on the hydrophilic side of the active center cavity, and one, FI98, is on the hydrophobic side. Recently these three residues in CAIII have been mutated to those that occur in CAll, with surprising results (39). The K64 � H change has essentially no effect on kca/Km. In contrast, the F 1 98L mutation increased kca/Km for the hydration of CO2 by ca. 20-fold, while the double mutant (FI98L, R67L) resulted in a 70-fold increase in kca/Km (kcat 2 x 104 S-I). Although kcatlKm for the double mutant shows little change on the additional mutation, K64H, to produce the triple mutant, there is an additional increase in kcat to 2 X 1 06 S- 1 , thus moving kcat for the triple mutant to that observed for CAlI. The most surprising finding was that all mutants containing the F I 98L substitution showed a pH activity profile with an apparent pKa of 6 . 8 t o 6 . 9 , very near to that of CAlI (39) . Thus , contrary to what might have been predicted, neither of the two charged side chains in CAllI significantly affects (i.e. lowers) the pKa of the coordinated solvent. Rather, it is the substitution =


904

COLEMAN

of one hydrophobic side chain (leucine) for another (phenylalanine) that affects both the pKa of the coordinated solvent and the rate of the reaction (39). The best guess as to the source of these effects is that the nature of the hydrophobic side chain in the cavity affects the precise arrangement of the hydrogen bond network connected to the Zn-OH2 or Zn- -OH. As with CAlI, the crystal structure of CAllI shows the Zn--OH or Zn-OH2 to be involved with the side chain of T l 99 and two H20 molecules which form a partially ordered network of at least nine hydrogen-bonded water molecules (39, 43 ) . I n CAllI one of these water molecules is observed to form a hydrogen bond with the 'IT" electron cloud of the F 1 98 ring. Thus the F 1 98L substitution may alter a hydrogen-bonded structure that is closely coupled to steps in the carbonic anhydrase mechanism and is primarily responsible for adjusting the pKa of the coordinated solvent molecule. In the past several years attention has been focused on the possibility that the solvent water molecule coordinated at the fourth position in many zinc metalloenzymes is a direct participant in their mech anisms as well . Carboxypeptidase A is one of the most interesting of these enzymes because it shows that although Zn may promote the attack of a coordinated solvent on a polarized carbon atom, there can be adjacent protein side chains that participate in both the polarization of the carbon atom and the formation of the incipient coordinated hydroxide species. The early crystal structure studies of carboxypeptidase A included the study of the structure of a complex with the slowly hydrolyzed dipeptide substrate, Gly-L-Tyr (44-46) . Significant features of the active center found at that time included the coordination of Zn to the N3 of H 1 96, the N3 of H69, and the carboxylate of E72, since shown to be bidentate ( 10, 47). A single coordi nated water molecule completed the zinc coordination sphere. The Gly-L-Tyr complex showed the presence of a hydrophobic pocket enclosing the aromatic side chain of the C-terminal residue, while its carboxyl group formed a salt bridge with R145 . These features are included in Figure 2, which is not of the Gly-L-Tyr complex, but indicates how a more normal substrate is thought to bind on the basis of recent crystallographic studies of transition-state analogs (reviewed in reference 48). The structure of the Gly-L-Tyr complex showed the carbonyl oxygen of the peptide bond to be coordinated to the Zn ion, displacing the coordinated H20. The placement of Gly-L-Tyr at the active site suggested that the carboxyl side chain of E270 participated either in a direct nucleophilic attack on the carbonyl carbon of the peptide bond or as a general base promoting the attack of an intervening water molecule on the carbonyl carbon (46) . The direct nucleophile alternative was favored and requires the formation of an anCARBOXYPEPTIDASE A


ZINC PROTEINS

905

hydride between the carboxyl of E270 and the new carboxyl of the hydrolysis product as an intermediate. More recent studies have shown that in the Gly-L-Tyr complex the free amino group and the carbonyl oxygen of the peptide group both coordinate the Zn, forming a chelate (48, 49). This nonproductive mode of binding is not available to typical substrates, in which the penultimate amino group is substituted. Coordination of the carbonyl oxygen adjacent to the coordinated H20 would probably hinder a hydrolysis reaction involving attack of the coordinated water on the carbonyl carbon, since the pKa of the former would be raised by the additional interaction of Zn with the carbonyl oxygen. An extensive series of cryogenic experiments demonstrated the existence of a series of metastable enzyme-bound in termediates during the hydrolysis of both esters and peptides by carboxypepti dase A (50-52). None of these intermediates react with trapping reagents, suggesting that none of the carboxypeptidase A reaction intermediates have the characteristics expected of the anhydride (50). The absence of an acyl enzyme intermediate, at least in the hydrolysis of peptides, was also sug gested by ISO-labeling experiments (53) . Recently there have been a series of crystallographic studies of the com plexes between the active center of craboxypeptidase A and substrate analog or inhibitor molecules , which appear to mimic the true substrate-binding mode or the transition state in a convincing manner (48 , 54). For example the inhibitor N-(tert-butoxycarbonyl)-5-amino-2-benzyl-4-oxo-6-phenylhexanoic acid is an extremely potent inhibitor of carboxypeptidase A (Kj 6.7 X 10 -7 M). This inhibitor is the ketonic analog of the substrate N-tert-boc-L phenylalanyl-L-phenylalanine. Like other nonactivated ketones, this com pound is hydrated at the carbonyl carbon at a level of less than 1 % in aqueous solution. Yet the crystal structure of the enzyme-inhibitor complex shows that the compound bound at the active center of carboxypeptidase A is present 100% as the hydrate. The enzyme appears to have favored the hydration reaction to achieve what is essentially the gem-diolate analog of the proteolyt ic tetrahedral intermediate (54). The polarization of one of the hydrate oxygens by both Zn2+ and Arg l 27+ stabilize it as the anion, the basis for proposing the intermediate pictured in Figure 2. The highest-resolution crystal structures of carboxypeptidase A show that upon the binding of inhibitors that coordinate the metal ion, the Zn moves slightly toward R127 along with a movement of En that alters its coordination toward a monodentate form (55 , 56) . As the pH of the crystalline enzyme is raised from pH 7.5 to 9 . 5 , the Zn-solvent bond decreases in length, possibly indicating an increase in the amount of the zinc hydroxide species (57). All of these rearrangements may be important aspects of substrate binding and catalysis. The compound N[[(benzyloxycarbonylamino]methyl]hydroxyphosphinyl L-Phe is the phosphonamidate analog of Cbz-Gly-Phe, one of the most rapidly =


906

COLEMAN

hydrolyzed peptide substrates for carboxypeptidase A (58). At pH 8 . 5 this phosphonamidate analog binds to carboxypeptidase A with the anionic moiety asymmetrically straddling the zinc ion (59). If the pH is lowered to 7 . 5 , the extra electron density found at the active center corresponds to the hydrolysis products of the phosphonamidate (60). Whether the enzyme actually partici pates in the hydrolysis is not clear, since the phosphonamidate linkage is already relatively unstable. Nevertheless, this mode of binding of a transition state analog also supports the notion that productive binding of a substrate is as illustrated in Figure 2. These crystal structures of reasonable transition state analogs support the mechanism of hydrolysis, as illustrated in Figures 2B and C. The Zn-coordinated water molecule is the nucleophile attacking the car bonyl carbon. The carboxyl of E270 not only activates this water by abstract ing a proton , but also is pictured as transferring a proton to the product in the final step. There has been discussion about whether Y248 or E270 is the proton transfer agent, and original references should be consulted. Thermolysin is a zinc peptidase from a thermophilic bacter ium; it has the same side chain specificity as carboxypeptidase A , but is an endopeptidase rather than a carboxy-exopeptidase. A complete summary of the extensive solution and crystal structure studies on this enzyme has been published by Matthews (61). The fact that the active centers of the two enzymes have similar coordination around the Zn ion [the N of H142, the N of H 1 46, the carboxylate of E166, and a coordinated H20 molecule (61-64)] is believed to indicate convergent evolution, since the amino acid sequences of the two enzymes are not related (65). The carboxylate of E 1 66 is a monodentate ligand in thermolysin, not a bidentate carboxylate like En of carboxypeptidase A (66). The positions and nature of the amino acid side chains at the active centers of carboxypeptidase A and thermolysin are so similar that their reaction mechanisms must be very similar if not identical (6 1 ) . E 1 43 of thermolysin is adjacent to the zinc site in a position to activate the coordinated water in the same manner as E270 does for carboxypeptidase A. Crystal structures of thermolysin-inhibitor complexes selected to represent a spectrum of analogs of the tetrahedral intermediate of peptide hydrolysis, as well as the reaction product carrying the free amino group, have been completed (summarized in reference 61) . From the structure of these com plexes, the solution kinetics , kinetic isotope effects, and 180 exchange data, the reaction mechanism pictured in Figure 3 has been proposed for thermo lysin (67). As with carboxypeptidase A, phosphonamidate inhibitors appear to provide reasonable transition state analogs (68). In thermolysin there is an additional stereochemical restriction on E143, which appears to preclude a direct nucleophilic attack of the carboxylate on the carbonyl carbon of a bound

THERMOLYSIN


A

-f?i\-

",,�� H, HIC-CO,::, � .I\,g-145 HN:I " HO-TY,248

/C'O_ _ Glu270 H,�.:-" 5.. 19 7

....

\

Figure 2

,,

6.:::-00: ,_

CH'"-�--@ c 0

196 72

His Glu·

50,197

�'

A�G+71

II o

'

I

--..'H"C-,O" O'-'- " ., \ H / / .... .... 'CH'-N-C in%· \ + 1/ / I \ .'g127 0,

TY,198/

Hjs Glu· His 196 72 69

' I

CH

C GIU270/ 'OH

-@ \

50,197\

CO

I

NH

+

.,g71

/

"

HC-CO/ =

o /I

H ' O-TY,248

'

�".�"'.

_

A'g145

= = = =

\ C O'" NH

c

Ty'I981

I

H zN

A,g1 - 45

/-@ /

"CH,-�-C ,

· 2:+ Zn

His/

'

= ==

' H,O-Ty,248

-O-C =: O \'"

/

Glu

19 6 72

0

II 0

\.

. .'g127 1.\HIS

"

69

\ •

A'g71

Mechanism of peptide hydrolysis proposed for the zinc exopeptidase carboxypeptidase A. (From reference 48.)

GJu/43 I . , ..

e p/e "

. HOH'

/

o

114

: 0 . .. .

}P .J!'sn 112

:.':

Z;,'

/1" (0)

'HN® ,

His 23/

I 114 ,113 ,e, r 0 o . / 0 ;!,snll2 H NH, H . ' ". !..... . 0 O· H i\ ",H H - C �C - N C� Q R, R, : 'e

.

/ 0 NH.

/'

Glu/43

Glul43

113

H H H i -C-C-N-CR, : U R;

Figure 3

//

HC-CO,-

/

/, \�� A,',;127

10 NH

Tyr198 /

,;"OH Tyr 157

I I Glu270" /'lH. :NH C "'-" '-I

__ = =

o II

�"'��"'�

B

Q

OW:P "HN@

/'

Tyrl57

His 231

I'

,

ii,

II" (bJ

I

G1u143,

C

U4

,1/3 ,C, eo'..0r ;!,snll2 o !

:

S

0 ./ .... . NH,

.

H !\ ®Hit H -C�C- N-C-

R: R, ···-;-i 'e OH · P······HN®

" Tyr/57

.'

ii'

II"

, fis231

¢>

114

o/e"'q

H

o

R,

'0

Tyrl57

(e)

Mechanism of peptide hydrolysis prop os ed for the zinc endopeptidase thermolysin. (From reference

Asnll2

.O;"NH,

N-C R,'

:

in'

/1' (d)

61.)

i

1/3

. .•.. ..�

®H;

I

-c-ce

",OH

o}-

HN:®

His 231

�

n

� �

2

en \0 o -.J

908

COLEMAN


peptide (69) . Hence the mixed anhydride mechanism was ruled out early. In the mechanism pictured in Figure 3 the Zn-coordinated water, activated by hydrogen bonding to E143, is the nucleophile attacking the carbonyl carbon of the scissile peptide bond. The proton abstracted by E143 could then be easily transferred to the leaving nitrogen. As indicated above, the dis advantage of the additional Zn-carbonyl oxygen interaction is a matter of discussion. ALCOHOL DEHYDROGENASE The mechanism of this enzyme, also with three protein ligands to the active site zinc and one solvent HzO molecule, illustrates another variation on zinc coordination chemistry involving monodentate ligands at the active centers (70). The oxidation-reduction catalyzed by alcohol dehydrogenase involves a typical hydride transfer to the nicotinamide ring of the NAD coenzyme. The precise role of the Zn in the mechanism of hydride transfer from the alcohol has been a matter of debate (70). Some proposals favor a mechanism in which the substrate binds to the coordinated water molecule rather than displacing the water, the latter acting as a general base. The alternative mechanism has the alcohol displace the water to coordinate directly as the alcoxide or at least significantly lower the pKa of the alcoholic -OR, facilitating the hydride transfer. There are a number of structural arguments , based on the crystal structure of the ternary complex with the substrate, which favor the alcoxide (7 1). The oxygen of the substrate is coordinated to the zinc, and the coordination sphere is hindered by the sulfur atoms such that water is excluded (see references 70 and 7 1 for discussion) . With regard to the induction of an alcoxide by zinc coordination, several lines of evidence now suggest that the phosphotransferase reaction catalyzed by alkaline phosphatase uses a similar species (see below). The alcohol or phenol acceptor, RzOH, in the R20H + R 10P032- - RZOP032+ R10H transferase reaction appears to coordinate the A-site zinc ion as the alcoxide, thus becoming the nucleophile instead of the -OH. The pH depen dence of the phosphotransferase activity relative to that of the phosphohydro lase activity supports this conclusion (see below).

Multi-Zinc Sites in Enzymes

Alkaline phosphatase is a dimeric zinc-contain ing phosphomonoesterase that is maximally active at alkaline pH and hydroly zes phosphate monoesters, ROPO/-. Alkaline phosphatase is widely distrib uted in mammalian tissues including intestines , kidneys , placenta, and bone. E. coli contains a similar enzyme inducible by phosphate starvation (72-74). With the proper adjustments for insertions or deletions, the amino acid sequences of several of the mammalian phosphatases can be fit to that of the E. coli enzyme (75). Such comparisons show that the essential activeALKALINE PHOSPHATASE


ZINC PROTEINS

909

center residues identified in the crystal structure of the bacterial enzyme are preserved in the eukaryotic enzymes. All alkaline phosphatases form a cova lent phosphoseryl intermediate on the reaction pathways, known as E-P (see below). The noncovalent complex with the product phosphate is termed E·P. In 1962 the enzyme from E. coli was shown to contain two zinc atoms per dimer (6). The presence of zinc in the mammalian alkaline phosphatases has since been confirmed in a number of studies (76). Following the initial report, the zinc: protein stoichiometry of the E. coli enzyme was revised upward to four or under some circumstances even six zinc atoms per dimer. The original lower stoichiometry occurred because the ammonium sulfate treatment used in the preparation readily removes zinc from one or two of the three metal binding sites now known to be present in each active center (77). If zinc is added to the preparation buffers, full stoichiometry can be achieved. In the presence of I to 10 mM Mg, two Zn ions and one Mg ion, rather than three Zn ions, are bound at each active center. Coordination chemistry at the active center of alkaline phosphatase The crystal structure of native zinc alkaline phosphatase (AP) from E. coli at 2-A

resolution shows that the active centers on each monomer contain three closely spaced metal-binding sites, two occupied by Zn ions and a third by an Mg ion (78). 113Cd nuclear magnetic resonance (NMR) of the I13Cd6 alkaline phosphatase dimer originally identified the presence of three Cd-binding sites in each monomer (79). Multinuclear NMR investigations of the enzyme have used the designations A, B , and C for the three sites; the crystal structure uses the designation 1 , 2, and 3. The "native" enzyme dimer has the metal composition (ZnAZnBMgchAP. The general polypeptide fold of the alkaline phosphatase monomer is shown in Figure 4. The active center is located at the carboxyl end of the central �-sheet, and all the ligands to the three metal ions are provided from one monomer. A computer graphics representation of the E· P complex of the native Zn4Mg2 enzyme, including the metal- ligand bonds, the slowly ex changing water molecules, the hydrogen bonds, and the amino acid side chains located within the immediate region of the active center, is shown in Figure 5. The Zn and Mg ions form a cluster in which the metal-to-metal distances trace a triangle of 3.94 by 4.88 by 7.09 A (79). Despite the close packing of the metal centers, there is only one bridging ligand, the carboxyl of AspS I , which bridges between Zn2 and Mg3 or Cd2 and Cd3 in the Cd6 enzyme. The A-site Zn has four ligands from the protein, which include both carboxyl oxygens ofD327, the N3 ofH331 and the N3 ofH412. In the absence of HPol-, water relaxation data (80) and 35Cl - NMR data

Zn}(A) coordination


910

COLEMAN

Figure 4 Structure of the monomer of alkaline phosphatase. The monomer-monomer interface of the dimer is on the left. The protein consists of a central lO-stranded f3-sheet flanked by 15 helices and another 3-stranded f3-sheet with a small helix at the top. (From reference 78.)

(8 1 ) suggest that the coordination sphere of the A-site Zn is completed by two H20 molecules. In the phosphate complex E'P , one of the phosphate oxygens forms a typical coordinate bond with Znl , with a Znl-O bond length of 1. 97 A and a Znl -0-P bond angle of 120° (78) . H372 , which was originally thought to coordinate Znl, is not a direct ligand (3 . 8 A away from Zn l ) , but the N3 of H372 is hydrogen bonded to one of the coordinated carboxyl oxygens of D327 . Zn2(B) coordination In the E'P complex Zn2 is coordinated tetrahedrally by the N3 of H370, one of the carboxyl oxygens of the bridging D5 1 , and one of the carboxyl oxygens of D369. The tetrahedral coordination is completed by a second phosphate oxygen, forming a phosphate "bridge" between Zn l and Zn2 . Although the Zn2-0 bond length is 1 . 97 A , identical to that for the Zn l -O bond, the Zn2-0-P bond angle at site B is nearly linear ( 1 75° ).


ZINC PROTEINS

911

Figure 5 The active-site region as it occurs in the E·P complex of alkaline phosphatase (E. coli) including all atoms within 10 A. of the bound phosphate. Water molecules are indicated by W, and hydrogen bonds are shown by the dashed lines. (From reference 78.)

The Mg or third Zn site can be described as a slightly distorted octahedron consisting of the second carboxyl oxygen of the bridging D5 1 , one of the carboxyl oxygens of E322 , and the hydroxyl of T155, with the rest of the coordination sites filled by three slowly exchanging water molecules. D153 is not a direct ligand as originally believed , but is an indirect ligand in that its carboxyl group forms hydrogen bonds with two coordinated water molecules, which are the direct ligands to Mg (Figure 5). The Mg site does not appear to be close enough to participate directly in the hydrolysis mechanism, but could of course make a contribution to the shape of the electrostatic potential around the active center.

Mg3(C) coordination

Enzyme-bound phosphate in the E·P imtermediate The structure of the E·P phosphoenzyme shows that two of the phosphate oxygens form oxygen-metal bonds to Znl and Zn2, while the other two form hydrogen bonds to the

912

COLEMAN

guanidinium group of R 1 66 (Figure 5) . The 31p NMR signal of E'P has provided the demonstration by saturation and inversion transfer that the dissociation of inorganic phosphate, kd 35 S-I, is the rate-limiting step in the mechanism at alkaline pH (82, 83) . Likewise the 31 p NMR signal of E-P has made possible the demonstration that the phosphoseryl residue in the apoenzyme is stable from pH 2 to 22 (84). Thus both phosphate binding and the dephosphorylation of the phosphoseryl intermediate are Zn dependent. E·P formed by the Zn4Mg2 enzyme has a 31p resonance at 4 ppm, whereas E-P resonates 8 ppm, downfield of phosphoric acid (77, 85) . The chemical shift of the E-P signal is relatively insensitive to the substitution of the various metal ion species at sites A and/or B, whereas the chemical shift of the E'P signal is highly sensitive to the nature of the metal ion in both sites: 3.4 ppm in Zn4Mg2 AP and 1 3 .0 ppm in Cd6AP. For the Cd6AP the 31p NMR signal suggested that the phosphate of E· P was coordinated to only one of the active-center Cd ions, since the 31p signal for E'P is a doublet 'showing a single 30-Hz 31P-113Cd coupling (77). Heteronuclear decoupling shows that this coupling comes from the A-site I13 Cd(II) , a conclusion supported by the disappearance of this coupling in the ZnACdB hybrid enzyme (86). Although the 31p coupling disappears upon Zn substitqtion at the A site, the unusual downfield chemical shift of the phosphorus resonance, 12.6 ppm, is main tained in the ZnACdB hybrid, leading to the unexpected conclusion that it is Cd at the B site rather than the A site that induces the unusual downfield shift of the E·31p signal . This became less surprising when the crystal structure of E'P showed the bound phosphate to bridge the two metal ions in sites A and l13CdB may be responsible for the absence of B. The unusual bonding to 31P_113Cd coupling. The electron density map of the active center in the absence of phosphate indicates a bond between the oxygen of S I02 and Zn2, supporting the notion that one of the functions of Zn2(B) is to deprotonate the Ser hydroxyl to SerO- . Although the 2-A map of the E' P complex shows this coordination position to be occupied by one of the phosphate oxygens and the side chain of S 102 appears free in the cavity (disordered) , S 102 may not be accessible to protons in the E· P complex. It is the metal ion in site A that controls the pH at which [E, P] [E-P], shifting from pH 5 for Zn in A site to pH 8 . 7 for Cd in A site (85, 87). This shift of ca. 3 pH units in the pKa controlling this eqUilibrium has sug gested that this equilibrium, which must reflect the apparent pKa of the activity, represents the proton dissociation from a coordinated water mole cule at the A site (83, 86). This is the most likely explanation for the metal dependent shift of ca. 3 pH units in this pKa as discussed above for carbonic anhydrase. If this is correct, Zn--OH becomes the nucleophile in the sec ond step of the hydrolysis mechanism attacking the phosphorus of E-P.


=

=

ZINC PROTEINS

913

Details of the many multinuclear NMR investigations of alkaline phosphatase have been reviewed previously (87, 88). The binding mode of the phosphate to the zinc ions is such that the oxygen of S 102 is in the required apical position to initiate a nucleophilic attack on the phosphorus nucleus (Figure 5). If this E· P structure is extrapolated to that of E·ROP, with which it must at least bear some features in common, the oxygen coordinated to ZnA must be the ester oxygen. None of the other positions would allow space for the R group of the substrate. This suggests that in the normal hydrolysis reaction, ZnA is an electrophile activating the leaving group, much as protonation of the ester oxygen docs in thc model systems. Thus it is possible that the alkaline phosphatase mechanism has as significant a dissociative component provided by the ZnA as it does an associative component provided by the attack of SerI02, the latter being activated by the second Zn ion. A dissociative character of the alkaline phosphatase reaction has been suggested by the low magnitude of secondary isotope effects when the nonbridging oxygens of the substrate are labeled with 180 (90). An electrophilic activation by the Zn would explain why there is not a significant difference in the (3 values for kcatlKm observed for substrates that cannot protonate the leaving group (phosphopyridines) compared with those that can (oxyesters) (89); both f3 values are equally small (89).


Michaelis complex of alkaline phosphatase with a phosphate monoester

Phosphoseryl intermediate

Phosphorylation of the Cd6 enzyme in solution and the crystal at pH 7.5 results in exclusive formation of the E-P intermediate (79). The electron density map of the resulting complex shows that a normal ester bond has formed with S 102 and that the phosphoryl group is slightly deeper in the active-center cavity than in the E·P complex. The phosphate oxygens remain very close to the metal ions, but the phosphate appears to have moved far enough from CdA to provide for the positioning of a coordin ated H20 COH) at CdA such that it can be the nucleophile attacking the phosphorus from the apical position opposite the seryl oxygen. There is a bond between the new ester oxygen of the phosphoseryl group and Cd2(B); thus the metal ion at site B appears to activate the leaving group in the second step of the mechanism in the same manner as the metal ion at site A does in the first step. Dissociation of inorganic phosphate from the positive center is then the final and slow step of the mechanism (82, 83). The two major hydrolysis steps of the mechanism are illustrated in Figure 6.

pl NUCLEASE The second zinc enzyme containing three zinc-binding sites in its active center is the endonuclease isolated from Penicillium citrinum,

914

COLEMAN

E·ROP

OH2

1� � 'PoH2

RO-

/

E-P


-=--_-�:>-R166 r:=::::> 2

Zn

ENZ

+

Pi

=-=--=->-R166 2

Figure 6

Mechanism of action proposed for alkaline phosphatase. The two half reactions

phosphorylation of Ser102 and the dephosphorylation of this covalent intermediate (E-P).

known as

PI

nuclease (92, 93). The crystal structure of

PI

nuclease at

are

2.8-11.

resolution shows that each active center contains three closely spaced Zn ions

with a bridging aspartate carboxyl between Zn l and Zn3, as well as a bridging water molecule or hydroxide ion (93). The enzyme is a phosphodiesterase which cleaves between the 3' -hydroxyl and the 5' -phosphoryl group of adjacent nucleotides. It also can act as a phosphomonoesterase and remove the

3' -terminal phosphate group from nucleotides (94). Both activities are

zinc dependent (95, 96). The enzyme belongs to a family of zinc-dependent nucleases that are isolated from several sources and show a preference for single-stranded nucleotides. Hydroly sis of the duplex nucleotides may be

associated with local melting of the substrate (93). Both DNA and RNA are hydrolyzed, although the phosphomonoesterase activity appears to be signifi cantly greater with

PI

RNA

substrates.

nuclease contains 270 amino acid residues (97). The amino acid s(�

quence shows the enzyme to be 50% homologous to the S I nuclease from Aspergillus oryzae (93), which has also been reported to require three zinc


ZINC PROTEINS

915

ions for activity (98). PI nuclease is largely helical , being made up of no less than 14 a-helices (Figure 7A). The three zinc ions of the active center are bound near the bottom of a large cleft formed between six of the helices . The Z n ions form a trinuclear site (Figure 7B). Zn l and Zn3 are c a . 3 . 2 A apart and are linked by a bridging carboxylate from D120 and by the oxygen of a water molecule or hydroxide ion (01, Figure 78). The third site , Zn2, is ca. 5 . 8 A from Zn l and 4. 7 A from Zn3 . Zn l is coordinated by the N-l of H60, the N-3 of H 1 l6, and the O-iH of D45 . Zn3 is coordinated by the N-I of H6, the nitrogen of the a-NH2 group of WI, the carbonyl oxygen of WI, and the other carboxylate oxygen of the bridging D120. Zn2 is coordinated by the N-3s of Hl26 and H149, the carboxylate of D 15 3 , and the oxygens of two solvent water molecules (0-2 and 0-3 , Figure 7B). Substrate interactions with the active center of PI nuclease Since the R-diastereomer of the thiophosphorylated dinucleotide, dA'P(S)'dA, in con trast to the S-isomer, is not hydrolyzed by PI nuclease, it was possible to soak the PI crystals in the R-isomer. The Fourier difference map shows two well-defined binding sites for the nucleotide ca. 20 A apart. Only one of these is near the trinuclear zinc complex. At the nucleotide-binding site near the Zn ions, one adenine ring is intercalated between the side chains of F61 and V32 (Figure 8). The carboxylate of D63 and the carbonyl oxygens of L25 and El28 are all in positions that could allow hydrogen bonds to form with the N6 of the bound adenine ring. The NI of the adenine could form a hydrogen bond with the carboxylate of D63 , but would require protonation of this side chain. The 3' half of the bound nucleotide is not visible in the difference map and is apparently disordered. The lack of density is not due to hydrolysis of the nucleotide, since no hydrolysis was detected over a period of weeks (93). A high peak of electron density near Zn2 has been interpreted as the phosphate ester group. The 5' ester oxygen and one of the unesterified phosphate oxygens are pictured as forming hydrogen bonds with the guanidinium group of R48. The other unesterified phosphate oxygen is within coordination distance of Zn2 and its two coordinated solvent molecules, 02 and 03. At the current stage of resolution of this "pseudosubstrate" complex there are several mechanistic possibilities. The phosphate oxygen could coordinate Zn2, displacing the 03 water ligand, with 02 becoming a Zn-coordinated nucleophile in the hydrolysis reaction. Alternatively, 03 could be the direct nucleophile attacking the adjacent phosphorus in the apical position to form the five-coordinate intermediate. This could be a Zn-OH2 or Zn- -OH. Volbe da et al (93) point out that the pH optimum for PI nuclease is between 4.5 and 6; they invoke several explanations for this , including the presence of two Asp-GIu pairs at the lip of the binding cavity, which would require the low pH for protonation. Otherwise the pairs would split apart, causing large con-


\0

B.

A.

; 36

Figure 7 (A) Crystal

:;

structure of Pl nuclease

nuclease. (From reference 93.)

(P. citrinum). (B)

Ligand structure of the three zinc complexes at the active center of Pl

ZINC PROTEINS


\

917

I I ""OS'

o-p

03

a

Figure 8 Diagram of the relationships of the thiophosphorylated dinucleotide dA-P(S)'dA bound at the active site of PI nuclease to the surrounding amino acids. This is the unhydrolyzed R isomer. (From reference 93.)

formational changes which might reduce activity. Muscle carbonic anhydrase (CAllI), however, has a pKa for the transition from the protonated (inactive) to the unprotonated (active) form of the enzyme below pH 5 , yet this pKa represents the Zn-OHz :;::: Zn- -OH + H equilibrium (42) (see above) . There fore an acidic pH optimum does not rule out the possibility that the pH activity profile reflects the ionization of a Zn-coordinated water molecule. PHOSPHOLIPASE c

The third enzyme discovered to possess a triad of Zn ions at its active center is phospholipase C from Bacillius cereus (99, 100) . The mammalian phospholipases C, which hydrolyze phosphatidylinositol and phosphatidylcholine, are catalysts in the pathways to the generation of second messengers which include inositol triphosphate (IP3) and diacylglycerol ( 1 0 1 , 102) . It was therefore of considerable interest to find in B. cereus an enzyme of 245 amino acids that catalyzed the hydrolysis of phosphatidylcholine and was similar to the mammalian phospholipases C ( 1 03). The functional sim ilarity was suggested when the B . cereus enzyme was found to enhance prostaglandin biosynthesis when added to the appropriate cell extract ( 1 04). Thus it may be a model for the poorly characterized mammalian enzymes. The crystal structure of the bacterial enzyme is available at 1 . 5-A. resolution as refined from 1 .9-A data (99) . This enzyme, like PI nuclease, is essentially


918

COLEMAN

an all-helix protein consisting of 10 helices rather than 14 (Figure 9A) . Much of its structure, however, can be overlaid on that of the PI nuclease. There is a cavity formed by parts of three helices , the N-terminal loop of the polypeptide, and one internal loop. At the bottom of this cleft are located three Zn ions with an almost identical arrangement and ligands to those of th(: three Zn sites found in PI nuclease. The structure of the Zn triad and its ligands in phospholipase C is summarized in Figure 9B . Because of the near identity to PI nuclease, a complete repetition of the details will not be given, but they include a bridging 0 1 22 carboxylate and bridging water or hydroxide between Znl and Zn3 and the coordination to Zn3 of the N-terminal W via both its amino group and its peptide carbonyl. The isolated Zn2 also carries two coordinated water molecules . The rest of the ligands are identical to those of PI nuclease. In contrast to the crystal structure, solution studies of phospholipase C have consistently indicated the presence of two zinc ions per molecule ( 100, 1 05 , 1 06) . There appeared to be two zinc sites that could be replaced with Cd, while extended X -ray absorption fine structure spectroscopy (EXAFS) of the Zn protein indicated a maximum of 2.3 Zn atoms, using a revised molecular weight (99, 107). Yet the crystal structure in the presence of 1 0 p, M free Zn(II) shows three bound Zn ions. This may be similar to alkaline phospha·· tase, for which preparation conditions in the absence of Zn buffers removed the metal ion from all but the highest-affinity binding site (see above).

A

Figure

9

(A) Crystal structure of phospholipase C. The positions of the three zinc ions are (B) Ligand structure surrounding the three zinc ions of phospholip. ase C. (From reference 99.) represented by the black balls.


ZINC PROTEINS

919

Studies with bound nonhydrolyzable analogs of the phosphodiester sub strate have not been done for phospholipase C, but a significant difference electrondensity map has been observed for the crystals soaked in phosphate (99) . Phosphate oxygens are coordinated to all three Zn ions, displacing the bridging water COR) between Znl and Zn3 and one of the water molecules from Zn2 . This is quite different from the relationships shown by the diester phosphate of the bound nucleotide in PI nuclease. Preliminary data, however, suggest that a similar complex is formed by the P I nuclease with inorganic phosphate (93). COMPARATIVE STRUCTURES OF THE ENZYMES CONTAINING MULTIPLE

Although native alkaline phosphatase contains two Zn sites and one Mg site, the enzyme is functional with three Zn ions and could be included among those with trinuclear Zn sites. Are the three enzymes with trinuclear Zn sites related? All three metal sites in PI nuclease and phospholi pase C are typical Zn-binding sites with mixed oxygen and nitrogen donors, whereas the third site in alkaline phosphatase is made up of only oxygen donors , which may explain its preference for Mg. The aspartate carboxylate bridging two of the Zn ions appears to define a binuclear zinc site in the two diesterases . In contrast, the bridging aspartate carboxylate defines the Zn-Mg pair in native alkaline phosphatase . Although all three enzymes are involved in phosphate ester hydrolysis, alkaline phosphatase has appeared to be exclusively a monoesterase , although one recent report has detected weak diesterase activity (1. F. Chlebowski, personal communication). There is only 1 8% amino acid sequence identity between the polypeptide chains of PI nuclease and phospholipase C, yet the crystal structures of the two enzymes show that 145 of their Ca atoms can be superimposed with a positional rms deviation of only 1 . 8 A. Thus PI nuclease and phospholipase C may well be evolutionarily related (93). The general polypeptide fold of the alkaline phosphatase monomer is clearly different from that of the other two enzymes (Figure 4) . In addition, alkaline phospha tase forms a phosphoseryl intermediate (Figure 6). Both PI nuclease and phosphlipase C appear to catalyze single-step hydrolyses with inversion of chirality around phosphate. For alkaline phosphatase the primary interaction of the monoester substrate is with the two Zn ions not linked by a bridging ligand. A second feature is the finding that the Zn ions coordinate the ester oxygens, activating the leaving groups in the initial scission of the R-O-P bond as well as in the hydrolysis of the phosphoseryl intermediate. The function of the isolated Mg or Zn site in the mechanism of action of alkaline phosphatase is less clear. The functions of the two Zn sites 3.9 A apart are obviously closely connected with the formation of the covalent intermediate. In contrast to alkaline phosphatase, the current information on substrate interactions with the diesterases PI nuclease and phospholipase C suggests ZINC SITES

920

COLEMAN

that the isolated Zn site, not the bridged binuclear site , is the site of primary interaction with the diester at least. This is also the site that appears to carry the potential Zn-OH2 or Zn--OH nuc1eophile (Figure 8). The lack of signifi cant interactions between the bound nucleotide and the binuclear Zn complex


in PI nuclease has led to the suggestion that the binuclear cluster might serve as an important structural element of the protein (93). The distribution within the polypeptide chain of the residues contributing the ligands to the zincs at the binuclear site is such that its formation ties together rather distant regions of the molecule. This may serve to stabilize the protein. Studies of the complexes of substrate analogs have not progressed as far for either the P I nuclease or the phospholipase C as they have for alkaline phosphatase , and the difference maps for phosphate bound to both PI nu clease and phospholipase C, which show that an interaction with inorganic phosphate can take place with all three zinc ions, raises the possibility that all three Zn ions can be catalytically involved at some stage of a complete mechanism. The simultaneous coordination of phosphate to more than one zinc relates these sites perhaps a little more closely to that of alkaline phosphatase, in which the phosphate of E'P bridges Zn l and Zn2. P I nuclease does have a phosphomonoesterase activity, and the mechanistic role of the three Zn ions could be different in the monoesterase and diesterase reactions. It may be premature to postulate exactly how similar or different the functions of the triad of metal ions at the active centers are in these three enzymes.

ZINC STORAGE AND TRANSPORT: ROLE OF METALLOTHIONEIN We know relatively little about the equilibria that must exist between storage sites in cells, free zinc in cell cytoplasm and body fluids , and the proteins in which zinc has a functional role. Despite the abundance of zinc in animal cells, little information is available on the true free zinc concentration in cells, i . e . the concentration of zinc not complexed to a macromolecule. Similarly, we do not know whether the free zinc concentration varies with time or whether significant zinc gradients develop between cell compartments or between cells. Large varations in the zinc content in different tissues ar{: observed ( 1 09) . This must mean at the very least that cell contents of zinc-containing macromolecules must vary. These differences are likely to represent other aspects of zinc metabolism as well, such as variation in th

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