TIBS16-MARCH1991
THE CARBOXY.TERMINAL a-amide group is a characteristic feature of many peptide hormones. In almost all cases, it is important for their bioactivity, and it contributes to their biostability. The demonstration of enzymecatalysed amidation was made possible by the use of peptide substrates stabilized against metabolic degradation; these allowed the formation of peptide amides to be observed in the presence of the multiplicity of proteases that occur in tissue extracts. The discovery that amidation is an enzyme-catalysed process ~ has led to extensive investigation of the enzymes that are involved and of the factors that regulate their catalytic activityz,3. Mechanlsmof amldatlon In the formation of peptide amides, the nitrogen atom of the amide is derived from the nitrogen of a mandatory carboxy-terminal glycine residue ~. It does not originate from ammonia as might have been expected, either by direct addition which occurs in the formation of glutamine from glutamic acid 4, or by ammonolysis of the peptide bond that links the glycine to the remainder of the peptide chain s. This surprising finding was demonstrated by observing that when a peptide with [~SN]glycineat its carboxyl terminus was converted to the corresponding des-glycine peptide amide, the product retained the ~SN-label (Fig. I). The production of [~4C]glyoxylate from the ~4C-labelled glycine residue showed that the amidation reaction was oxidative, a conclusion that is supported by the observation that ascorbate ~ and catechols such as dopamine 8 can act as cofactors. The oxygen involved in the reaction appears to originate from molecular oxygen, since amidation is inhibited under anaerobic conditions 7. As with many oxidizing enzymes, the amidati.n_g enzyme has been shown to have a mandatory and specific requirement for copper 7-9. However, the metal is only loosely held to the protein: it can be removed by dialysis against EDTA, with loss of activity, and the activity is restored on replacement of the copper ~°. The requirement for copA. F. Bradburyand D. G. Smythare at the National Institutefor Medical Research,The Ridgeway,Mill Hill, LondonNW7 1AA,UK. 112
Many hormones, neurotransmitters and growth factors are peptides that carry an amide group at their carboxyl terminus which is essential for their biological activity. The amide is formed by hydroxylation of an additional glycine residue present in the biosynthetic precursor and the hydroxyglycine derivative dissociates to form the peptide amide and glyoxylic acid. Recent discoveries have shown that two enzymes are involved that act sequentially.
per and oxygen and the stimulation by ascorbate suggests a similarity with another oxidizing enzyme, dopamine hydroxylase. This seems to be justified since the molecular environment of the copper seems to be similar in the two enzymes H and their primary sequences indicate a common evolutionary origin 12. That the amidation reaction involves an initial hydroxylation step s was first shown with the use of a non-peptide substrate, glyoxylic acid phenylhydrazone ~3. The product formed from the hydrazone rearranged to oxalic acid phenylhydrazide, a stable compound that was readily identified (Fig. 2). Very recently, a hydroxylated intermediate formed during the amldatlon of a peptide has been detected 14 and its structure firmly established ~s.It has been suggested that the enzyme that catalyses the oxidative stage of the amidation reaction should be called 'peptidylglycine hydroxylase 'z,~a. The second stage in the amidation reaction involves conversion of the hydroxyglycine intermediate to the peptide amide and glyoxylic acid. This takes place without enzymic assistance at acid or alkaline pH, but appears to occur relatively slowly at physiological pH. An interesting recent finding is that an enzyme exists that will hasten the conversion of the hydroxylated intermediate 14-~s and it is probably significant that this enzyme occurs in the same tissues as the hydroxylase. Amidation in vivo is thus seen to be a two-stage process catalysed by different enzymes (E~ and E2 in Fig. I). It has been suggested that the second enzyme should be named peptidylhydroxyglycine N--Clyase Is. Previous reports ~9of a high pH optimum for enzymic amidation (in the region of 8.5-9.09) may be explained by
a form of 'compromise' between the optimum pH of the hydroxylase (6.5-7.0) and the higher pH that is necessary for non-enzymic breakdown of the hydroxylated intermediate. Indeed, a recent report z° described a 41 kDa protein that co-localized with the 'amidating' enzyme but was devoid of amidating activity; this protein could lower the apparent pH optimum of the amidating reaction. It is likely that this protein is in fact the second enzyme. It may be that the purpose of the second enzyme is not only to speed up the formation of the amide, but also to ensure that the release of glyoxylate occurs at a site in the cell where such a reactive molecule would be relatively harmless. In this review, the term 'amidatlng enzyme', which has been widely used in the literature, refers to enzyme preparations that contain both the hydroxylase and the N--C lyase. Little is known yet of the structure, mechanism and properties of the lyase, which accelerates the breakdown of the hydroxylated intermediate to form peptide amide and glyoxylate.
Distribution The first studies on enzyme-catalysed amidation were carried out with enzyme obtained from pituitary, because it was known that the amidated hormones oxytocin, vasopressin and a-melanocyte-stimulating hormone (oc-MSH), occur in this tissue. Subsequent studies, however, have demonstrated that amidating activity is widely distributed: it is present in almost every tissue including heart, thyroid, hypothalamus, adrenal medulla, submandibular gland, pancreas, intestine, bone, prostate and seminal fluid. Amidating activity also occurs in serum and it is interesting that the circulating levels are not significantly reduced by
© 1991,ElsevierSciencePublishersLtd,(UK) 0376-5067/91/$02.00
TIBS 16-MARCH1991 hypophysectomy (removal of the pituitary gland) '-~.It appears, therefore, that the amidating enzyme or enzymes in the blood originate from a variety of different tissues. Experiments with cultured cells have shown that amidated hormones are secreted together with the enzymes that catalyse their formation2z.This suggests that the amidating enzyme in serum arises as a byproduct of processing events that have taken place intracellularly prior to secretion. It is not known whether the same conclusion can be drawn on the much higher levels of amidating activity that occur in semen and in this case the possibility of extracellular amidation should not be excluded. Since a variety of different amino acids are found at the carboxyl terminus of hormone amides and the rate of amidation is dependent on the nature of the residue undergoing amidation ~,24, there was a possibility that a number of different enzymes having preferential speciflcities for different substrates might exist. This now appears unlikely, since the relative rates of amidation observed with synthetic substrates corresponding to the carboxy-terminal sequences of oxytocin, gastrin, vasoactive intestinal peptide (VIP) and a-MSH have been found to be the same with amidatlng enzyme obtained from different tissues ~,2s. Amldating enzymes and amidated peptldes occur in a wide variety of species, ranging from anglerfish to insects and mammals, and it is likely that the amide group is formed by the same mechanism in every case. For example, the amidated bee-venom peptide, mellitin, is known to originate from a precursor that contains an additional carboxy-terminal glycine ~7, which is characteristic of the mam~,lalian amidation mechanism; this is consistent with the view that the hydroxylating mechanism is universally applicable.
more recently from rat3~,32 and man33, complete amino acid sequences of a number of preproenzymes have been derived and it is notable that certain species, such as Xenopus and rat, elaborate more than one preproenzyme (Fig. 3). Notably, all the sequences include an approximately 40 kDa domain, and because this core region accounts for the whole of one of the Xenopus cDNA sequences it can be presumed to contain the active site of the enzyme. Indeed, the smallest size of active enzyme that has been isolated possesses a molecular weight in the region of 40 kDa9,~,~. Other features apparent from the cDNA sequences of the larger enzymes are the presence of a putative transmembrane domain, potential sites for carbohydrate attachment and the occurrence of paired basic amino acid residues which offer possible cleavage points for proteolytic processing enzymes. Since these structural features occur outside the 'active core', it appears that none are essential for the fundamental activity of the enzyme. While proteolytic cleavage at paired basic amino acid sites seems likely to account for much of the observed heterogeneity, the functional significance of the different forms remains unexplained. Similarly, various forms of the enzyme exist that differ in their carbohydrate content 3s, the function of which is unknown. The strongly hydrophobic region, which occurs near the carboxyl terminus of the II0 kDa molecule, might play a role in targetting this enzyme into the secretory granule; alternatively, it could fulfil a role in immobilizing the enzyme within the granule. However, most of the enzyme can be extracted readily from secretory granules without the use of a detergent, which indicates that once in the granule, the majority of the enzyme is present in a soluble form, or is only loosely attached to the membranes. In contrast, in the rough endoplasmic reticuMultiple enzymeforms lum (PER) a higher proportion of the When extracts of pituitary are frac- enzyme occurs in a membrane-bound tionated by gel exclusion chromatography, form37. the 'amidating enzyme' behaves as if it were present in a number of forms that Physiological regulation It is perhaps surprising that the indiffer in molecular size ~. This might be explained by the existence of multiple active glycine-extended precursors of proenzymes or the different size some amidated hormones occur in enzymes might be generated from a higher concentrations than their biosingle precursor. With the determination logically active amidated counterparts 38. of the cDNA sequence of amidating Consequently, increasing the amidating enzymes ~.pti~ylglycine hydroxyl- activity in a cell might lead to an ases) from Xenopus~s.~9 and ox 3°, and increase in the amount of hormone
Peptide-COlSNH.CH2.CO2H
Peptide-COlSNH.CH.CO2H
I
OH
Peptide-COlSNH2 + CH.CO2H
II
0 Figure 1 Mechanism of enzyme-catalysedpeptide amidation1.
amide generated, and this could represent one level at which physiological regulation occurs. Regulation of amidation may take place at the gene level during post-translational processing, or by variation in the availability of cofactors. It is also possible that regulation might take place by control of access of the amidating enzyme to its substrate. Experiments have shown that amidating activity can be influenced by a variety of hormonal, drug and dietary factors 38. It has been reported 4° that depriving AtT-20 mouse pituitary tumour cells of copper by chronic treatment with disulphiram, a copper chelator, led to a reduction in the degree of amidation of the 'hinge' or 'joining' peptide, the peptide that links adrenocorticotropic hormone (ACTH) to the aminoterminal region of the ACTH-endorphin prohormone. Chronic treatment of rats
Ph.NH.N=CH.CO2H
Ph.NH.N=C.CO2H
I OH
Ph.NH.NH-C.CO2H
II 0
Rgure 2 Oxidation of glyoxylic acid phenylhydrazone to oxalic acid phenylhydrazidecatalysed by peptidyl glycine hydroxylase13. 113
TIBS 16-MARCH1991
preproenzyme residue number
0
100
200
300
400
I
I
I
I
I
500
600
I
I
700
I
800
900
1000
I
I
I
NH 2
I
Type A
NH 2
Type B
B/
I
c
c
I
Ii
.=
4
El il !!1
Active core
Rgure 3 Diagrammatic representation of amidating enzymes predicted by translation of cDNA sequences derived from a rat thyroid cDNA library by Bertelsen et al. 31 The signal sequences are indicated by the hatched areas, the amino terminus of the enzyme by NH2, putative transmembrane domains by the solid areas, potential glycosylation sites by C, and paired basic amino acid residues offering possible cleavage sites by the vertical dashed lines. The two predicted enzymes (Type A and Type B) appear to be formed by alternative gene splicing.
with disulphiram reduced the pituitary levels of both the 'hinge' peptide and MSH39. Similarly, depletion of ascorbate in cultured pars intermedia cells led to a reduction in the concentration of the amidated form of a-MSH relative to its glycine-extended precursoP L42.Anterior pituitary cells showed a similar effect: the amidating activity declined when ascorbate was removed from the culture media but partiaUy recovered when the levels were restored 4~. in the case of the guinea pig, which is unable to synthesize ascorbate, depletion in dietary levels of this potential cofactor can reduce the extent of amidation of pituitary (I-MSH43. However, it should be noted that the removal of copper or ascorbate may have wide ranging consequences apart from a possible influence on amidation, and it has not been established unequivocally that either copper or ascorbate is actually involved in the normal physiological regulation of amidation. Recently, 4-phenyi-3-butenoic acid was shown to be an in vivo inhibitor of peptidylglycine hydroxylase 44. It was found to bring about a reduction in the intracellular amidating activity in CA77 rat thyroid carcinoma cells and interestingly the results pointed to the existence of a homeostatic mechanism used by the cells for maintaining levels of amidating activity. The cells appear to respond to the presence of the inhibitor by producing more enzyme. In the same way, administration of diethyldithiocarbamate (DDC) to rats led to decreased production of gastrinamide and this was followed by an increase in ami114
surface protein. This approach might, for example, allow the amidation of gastrin to be controlled, which may be of value in the treatment of peptic ulcers. Furthermore, the application of an inhibitor could open the way to studying whether the enzyme fulfils a function in the cell in addition to peptide amidation. Since only two of the enzymes involved in mammalian prohormone processing have been positively identified, of which peptidylglycine hydroxylase is one, studies of the biosynthesis, intracellular transport and processing of this enzyme should greatly increase our understanding of how cells produce and control the activity of peptide hormones.
References 1 Bradbu~y,A. F., Rnnie, M. D. A. and Smyth, D. G. (1982) Nature 298, 686-688 dating enzyme synthesis 45. Also, glu- 2 Bradbury,A. F. and Smyth, D. G. (1987) Biosci. Rep. 7, 907-916 tathione, a tripeptide that is present in 3 Eipper, B. A. and Mains, R. E. (1988) Annu. most cells and possesses a carboxyRev. Physiol. 50, 333-344 terminal glycine, would be capable of 4 Meister, A. (1962) in The Enzymes (Vol. 6), pp. 443-445 competing in the amidation reaction by 5 Fruton, J. S., Johnston, R. B. and Fried, M. binding to the amidating enzyme. If this (1951) 1 Biol. Chem. 190, 39-53 were to occur, less enzyme would be 6 Bateman, R. C., Youngblood,W. W., Busby, available for the amidation of other W. H. and Kiser, J. S. (1985) J. BioL Chem. 260, 9088-9091 peptides. 7 Eipper, B. A., Mains, R. E. and Glembotski, C. C. (1983) Proc. Natl Acad. Sci. USA 80, Future directions 5144-5148 High-affinity antibodies have now 8 Landymore-Um,A., Bradbury, A. F. and Smyth, D, G. (1983) Biochem. Biophys. Res. been raised to synthetic peptides that Commun. 117, 289-293 correspond to regions of the sequence 9 Mizuno, K., Sakata, J., Kojima, M., Kangawa, K. of peptidylglyclne hydroxylase. This and Matsuo, H. (1986) Biochem. Biophys. Res. Commun. 137, 984-991 should allow immunocytochemical methods to be used for tracing the 10 Bradbury, A. F. and Smyth, D. G. (1985) in Biogenetics of Neurohormonal Peptides route of the enzyme from its biosyn(H~kanson, R. and Theorell, J., eds), thesis in the rough endoplasmic reticupp. 171-186, Academic Press lum to its appearance in the secretory 11 Markoslan, K, A., Paitian, N. A., Abramian, K. S. and Nalbandian, R. M. (1989) Biokhimicia 54, granule and ultimately to its secretion 2046-2053 from the cell surface. Since glycine- 12 Southan, C. and Kruse, L. I. (1989) FEBS Lett. extended precursors can occur in sig255, 116-120 nificant quantities 38, the question of 13 Bradbury, A. F. and Smyth, D. G. (1987) Eur. J. Biochem. 169, 579-584 where the substrate and amidating 14 Young, S. D. and Tamburini, P. P. (1989) J. Am. enzyme are brought together arises. Chem. Soc. 111, 1933-1934 Are they initially located in separate 15 Suzuki, J., Shimoi, H., Iwasaki, Y., Kawahara, T., Matsuura, Y. and Nishikawa, Y. (1990) EMBO J. compartments that undergo fusion 13, 4511-4517 before amidation takes place, or are the 16 Katopodis, A. G., Ping, D. and May, S. W. (1990) amidating enzyme and its substrates Biochemistry 29, 6116-6120 always present in the same compart- 17 Takahashi, K., Okamoto, H., Seino, H. and Noguchi, M. (1990) Biochem. Biophys. Res. ment, with the extent of amidation Commun. 169, 524-530 being a function of time and depending 18 Tajima, M., lida, T., Yoshida, S., Komatsu, K., on the availability of cofa(.~ors? Namba, R., Yanagi, M., Noguchi, M. and Okamoto, H. (1990) J. Biol. Chem. 265, The development of specific inhibitors of peptidylglycine hydroxyl- 19 9602-9605 Murthy, A. S. N., Mains, R. E. and Eipper, B. A. ase that penetrate cells raises the possi(1986) J. Biol. Chem. 261, 1815-1822 bility that the inhibitor might be targetted 20 Noguchi, M., Takahashi, K. and Okamoto, H. (1989) Arch. Biochem. Biophys. 275, to a tissue by attachment to an appro505-513 priate 'address' molecule, such as an 21 Eipper, B. A., Myers, A. C. and Mains, R. E. antibody with affinity for a specific cell(1985) Endocrinology 116, 2497-2504
TIBS16- MARCH1991 22 Mains, R. E. and Eippar, B, A. (1984) Endocrinology 115,1683-1690 23 Bradbury, A. F. and Smyth, D. G. (1983) Biochem. Biophys. Res. Commun. 112, 372-377 24 Tamburini, P. P., Jones, B. N., Consalvo, A.P., Young, S. D., Lovato, S. J., Gilligan, J.P., Wennogle, L. P., Erion, M. and Jeng, A. Y. (1988) Arch. Biochem. Biophys. 267, 623-631 25 Bradbury, A. E and Smyth, D. G. (1983) in Peptides, Structure and Function (Hruby, V. J. and Rich, D. H., eds), pp. 249-253, Pierce Chemical Company 26 Tamburini, R R, Young, S. D., Jones, B. N., Palmesino, R. A. and Consalvo, A. R (1990~ Int. J. Pept. Protein Res. 35,153-156 27 Suchanek, G. and Kreil, G. (1977) Proc~Natl Acad. Sci. USA 74, 975-978 28 Mizuno, K., Ohsuye, K., Wada, Y., Fuchimura, K., Tanaka, S. and Matsuo, H, (1987) Biochem. Biophys. Res. Commun. 148, 546-553 29 Ohsuye, K., Kitano, K., Wada, Y., Fuchimura, K.,
Tanaka, S., Mizuno, K. and Matsuo, H. (1968) Biochem. Biophys. Res. Commun. 150, 1275-1281 30 Eipper, B. A., Park, L. P., Dickerson, I. M., Keutmann, H. T., Thiele, E. A., Roddguez, H., Schofield, P. R. and Mains, R. E. ~1987) Mol. Endocrinol. 1, 777-790 31 Birtelsen, A. H., Beaudry,G. A., Galella, E. A., Jones, B. N., Ray,M, L. and Matea, N. M. (1990) Arch. Biochem. Biophys. 279, 87--96 32 Stoffers, D. A., Green, C. B-R. and Eipper, B. A. (1989) Proc. Natl Acad. Sci. USA 86, 735-739 33 Glauder, J., Ragg, H., Rauch, J. and Engels, J. W. (1990) Biochem. Biophys. Res. Commun. 169, 551-558 34 Murthy, A. S. N., Mains, R. E. and Eipper, B. A. (1986) J. BioL Chem. 261,1815-1822 35 Bradbury, A. F. and Smyth, D. G. (1988)in Progress in Endocrinology(Imura, H., Shizume, K. and Yoshida, S., eds), pp. 335-340, Elsevier Science Publishers 36 Bradbury, A. E and Smyth, D. G. (1988)
Biochem. Biophys. Res. Commun. 154, 1293-1300 37 May, V., Cullen, E. I., Braas, K. M. and Eipper, B. A. (1988) J, BioL Chem. 263, 7550-7554 38 Sugano, K., Park, J., Dobbins, W. D. and Yamada, J. (1987) Am. J. Physiol. 253, 6502-6507 39 Mains, R. E., Myers, A. C. and Eigper, B. A. (1985) Endocrinology 116, 2505-2515 40 Mains, R. E., Park, L. R and Eipper, B. A. (1986) J. Biol. Chem. 261,11938-11941 41 May, V. and Eipper, B. A. (1985) J. Biol. Chem. 260,16224-16231 42 Glembotski, C. C. (1986) Endocrinology 118, 1461-1468 43 Fenger, M. and Hilsted, L. (1988) Acta EndocrinoL 118, 119-125 44 Bradbury, A. F., Mistry, J., Roos, B. A. and Smyth, D. G. (1990) Eur. J. Biochem. 189, 363-368 45 Dickinson, C. J., Marino, L. and Yamada,T. (1990) Am. J. PhysioL 285, 6810-6814
REFLECTIONS onBIOCHEMISTRY DESPITE 135 RECENT downturn, the wool industry is of immense importance to the Australian economy, and Australians still talk of 'living off the sheep's back'. There are, in fact, roughly ten sheep for every person living in Australia. However, since World War II, wool has been steadily losing its market share to man-made fibres, until it now retains only 5% of the world textile market. Since the invention of nylon by Carothers in 1936, the threat of the man-made fibre industry to wool has been recognized by the Australian wool industry, so that in 1948 the Government appointed an expert committee to suggest ways to improve the utilization of wool. As a result of recommendations by this committee, the Wool Textile Research Laboratories were established within Australia's premier research body, the Commonwealth Scientific and Industrial Research Organization (CSIRO). One of these, the Parkville (Victoria) laboratory, was established to investigate the chemistry of the wool fibre. In 1959 this laboratory became the CSIRO Division of Protein Chemistry (DPC), which was responsible for much of the research on
Wool, a dead tissue of epithelial origin, derives many of its properties as a textile fibre from the structure and arrangement of the proteins from which it is comprised. Much of the progress in the elucidation of wool protein structures, as a step towards understanding this relationship between structure and properties, has been made in the Division of Protein Chemistry of Australia's Commonwealth Scientific and Industrial Research Organization.
the proteins of wool. The fundamental research on wool proteins at its zenith occupied some 13-14 of the 55 professional scientists on the staff at the DPC. Until the wool industry suffered a serious setback in the late 1960s and early 1970s, the DPC was almost entirely concerned with various aspects of wool chemistry, but after this time diversified its activities into other areas of biochemistry. The work of the DPC was at least partly responsible for Nobel Laureate Dr Arthur Kornberg stating in 1968, in a plea for support for biological research in the USA, ' . . . we know less about the molecul~s of our chromosomes and our brain than we do L. M. Dowllngand L. 6. Span~w are at CSIRO, about the molecules in Austra!i~n wool. Division of WoolTechnology,343 Royal This is because Australian wool growParade,Parkville,Victoria3052, Australia.
© 1991,ElsevierSciencePublishersLtd,(UK) 0376-5067/91/$02.00
ers have for many years recognized the value of basic research in the composition and chemical properties of wool.' It is an interesting coincidence that this basic wool research has revealed structures common to both wool and cells of the brain. The funds to support this research come partly from direct contributions from woolgrowers and are administered by the Wool Research Committee on which woolgrowers have a strong representation. Humans use wool for much the same purpose as does the sheep - for protection, warmth and decoration. These are all mechanical and structural uses rather than dynamic ones, so the prime study of wool is one of structure. Wool is a dead tissue of epithelial origin, and is classified as a hard a-keratin along
115
THE CARBOXY.TERMINAL a-amide group is a characteristic feature of many peptide hormones. In almost all cases, it is important for their bioactivity, and it contributes to their biostability. The demonstration of enzymecatalysed amidation was made possible by the use of peptide substrates stabilized against metabolic degradation; these allowed the formation of peptide amides to be observed in the presence of the multiplicity of proteases that occur in tissue extracts. The discovery that amidation is an enzyme-catalysed process ~ has led to extensive investigation of the enzymes that are involved and of the factors that regulate their catalytic activityz,3. Mechanlsmof amldatlon In the formation of peptide amides, the nitrogen atom of the amide is derived from the nitrogen of a mandatory carboxy-terminal glycine residue ~. It does not originate from ammonia as might have been expected, either by direct addition which occurs in the formation of glutamine from glutamic acid 4, or by ammonolysis of the peptide bond that links the glycine to the remainder of the peptide chain s. This surprising finding was demonstrated by observing that when a peptide with [~SN]glycineat its carboxyl terminus was converted to the corresponding des-glycine peptide amide, the product retained the ~SN-label (Fig. I). The production of [~4C]glyoxylate from the ~4C-labelled glycine residue showed that the amidation reaction was oxidative, a conclusion that is supported by the observation that ascorbate ~ and catechols such as dopamine 8 can act as cofactors. The oxygen involved in the reaction appears to originate from molecular oxygen, since amidation is inhibited under anaerobic conditions 7. As with many oxidizing enzymes, the amidati.n_g enzyme has been shown to have a mandatory and specific requirement for copper 7-9. However, the metal is only loosely held to the protein: it can be removed by dialysis against EDTA, with loss of activity, and the activity is restored on replacement of the copper ~°. The requirement for copA. F. Bradburyand D. G. Smythare at the National Institutefor Medical Research,The Ridgeway,Mill Hill, LondonNW7 1AA,UK. 112
Many hormones, neurotransmitters and growth factors are peptides that carry an amide group at their carboxyl terminus which is essential for their biological activity. The amide is formed by hydroxylation of an additional glycine residue present in the biosynthetic precursor and the hydroxyglycine derivative dissociates to form the peptide amide and glyoxylic acid. Recent discoveries have shown that two enzymes are involved that act sequentially.
per and oxygen and the stimulation by ascorbate suggests a similarity with another oxidizing enzyme, dopamine hydroxylase. This seems to be justified since the molecular environment of the copper seems to be similar in the two enzymes H and their primary sequences indicate a common evolutionary origin 12. That the amidation reaction involves an initial hydroxylation step s was first shown with the use of a non-peptide substrate, glyoxylic acid phenylhydrazone ~3. The product formed from the hydrazone rearranged to oxalic acid phenylhydrazide, a stable compound that was readily identified (Fig. 2). Very recently, a hydroxylated intermediate formed during the amldatlon of a peptide has been detected 14 and its structure firmly established ~s.It has been suggested that the enzyme that catalyses the oxidative stage of the amidation reaction should be called 'peptidylglycine hydroxylase 'z,~a. The second stage in the amidation reaction involves conversion of the hydroxyglycine intermediate to the peptide amide and glyoxylic acid. This takes place without enzymic assistance at acid or alkaline pH, but appears to occur relatively slowly at physiological pH. An interesting recent finding is that an enzyme exists that will hasten the conversion of the hydroxylated intermediate 14-~s and it is probably significant that this enzyme occurs in the same tissues as the hydroxylase. Amidation in vivo is thus seen to be a two-stage process catalysed by different enzymes (E~ and E2 in Fig. I). It has been suggested that the second enzyme should be named peptidylhydroxyglycine N--Clyase Is. Previous reports ~9of a high pH optimum for enzymic amidation (in the region of 8.5-9.09) may be explained by
a form of 'compromise' between the optimum pH of the hydroxylase (6.5-7.0) and the higher pH that is necessary for non-enzymic breakdown of the hydroxylated intermediate. Indeed, a recent report z° described a 41 kDa protein that co-localized with the 'amidating' enzyme but was devoid of amidating activity; this protein could lower the apparent pH optimum of the amidating reaction. It is likely that this protein is in fact the second enzyme. It may be that the purpose of the second enzyme is not only to speed up the formation of the amide, but also to ensure that the release of glyoxylate occurs at a site in the cell where such a reactive molecule would be relatively harmless. In this review, the term 'amidatlng enzyme', which has been widely used in the literature, refers to enzyme preparations that contain both the hydroxylase and the N--C lyase. Little is known yet of the structure, mechanism and properties of the lyase, which accelerates the breakdown of the hydroxylated intermediate to form peptide amide and glyoxylate.
Distribution The first studies on enzyme-catalysed amidation were carried out with enzyme obtained from pituitary, because it was known that the amidated hormones oxytocin, vasopressin and a-melanocyte-stimulating hormone (oc-MSH), occur in this tissue. Subsequent studies, however, have demonstrated that amidating activity is widely distributed: it is present in almost every tissue including heart, thyroid, hypothalamus, adrenal medulla, submandibular gland, pancreas, intestine, bone, prostate and seminal fluid. Amidating activity also occurs in serum and it is interesting that the circulating levels are not significantly reduced by
© 1991,ElsevierSciencePublishersLtd,(UK) 0376-5067/91/$02.00
TIBS 16-MARCH1991 hypophysectomy (removal of the pituitary gland) '-~.It appears, therefore, that the amidating enzyme or enzymes in the blood originate from a variety of different tissues. Experiments with cultured cells have shown that amidated hormones are secreted together with the enzymes that catalyse their formation2z.This suggests that the amidating enzyme in serum arises as a byproduct of processing events that have taken place intracellularly prior to secretion. It is not known whether the same conclusion can be drawn on the much higher levels of amidating activity that occur in semen and in this case the possibility of extracellular amidation should not be excluded. Since a variety of different amino acids are found at the carboxyl terminus of hormone amides and the rate of amidation is dependent on the nature of the residue undergoing amidation ~,24, there was a possibility that a number of different enzymes having preferential speciflcities for different substrates might exist. This now appears unlikely, since the relative rates of amidation observed with synthetic substrates corresponding to the carboxy-terminal sequences of oxytocin, gastrin, vasoactive intestinal peptide (VIP) and a-MSH have been found to be the same with amidatlng enzyme obtained from different tissues ~,2s. Amldating enzymes and amidated peptldes occur in a wide variety of species, ranging from anglerfish to insects and mammals, and it is likely that the amide group is formed by the same mechanism in every case. For example, the amidated bee-venom peptide, mellitin, is known to originate from a precursor that contains an additional carboxy-terminal glycine ~7, which is characteristic of the mam~,lalian amidation mechanism; this is consistent with the view that the hydroxylating mechanism is universally applicable.
more recently from rat3~,32 and man33, complete amino acid sequences of a number of preproenzymes have been derived and it is notable that certain species, such as Xenopus and rat, elaborate more than one preproenzyme (Fig. 3). Notably, all the sequences include an approximately 40 kDa domain, and because this core region accounts for the whole of one of the Xenopus cDNA sequences it can be presumed to contain the active site of the enzyme. Indeed, the smallest size of active enzyme that has been isolated possesses a molecular weight in the region of 40 kDa9,~,~. Other features apparent from the cDNA sequences of the larger enzymes are the presence of a putative transmembrane domain, potential sites for carbohydrate attachment and the occurrence of paired basic amino acid residues which offer possible cleavage points for proteolytic processing enzymes. Since these structural features occur outside the 'active core', it appears that none are essential for the fundamental activity of the enzyme. While proteolytic cleavage at paired basic amino acid sites seems likely to account for much of the observed heterogeneity, the functional significance of the different forms remains unexplained. Similarly, various forms of the enzyme exist that differ in their carbohydrate content 3s, the function of which is unknown. The strongly hydrophobic region, which occurs near the carboxyl terminus of the II0 kDa molecule, might play a role in targetting this enzyme into the secretory granule; alternatively, it could fulfil a role in immobilizing the enzyme within the granule. However, most of the enzyme can be extracted readily from secretory granules without the use of a detergent, which indicates that once in the granule, the majority of the enzyme is present in a soluble form, or is only loosely attached to the membranes. In contrast, in the rough endoplasmic reticuMultiple enzymeforms lum (PER) a higher proportion of the When extracts of pituitary are frac- enzyme occurs in a membrane-bound tionated by gel exclusion chromatography, form37. the 'amidating enzyme' behaves as if it were present in a number of forms that Physiological regulation It is perhaps surprising that the indiffer in molecular size ~. This might be explained by the existence of multiple active glycine-extended precursors of proenzymes or the different size some amidated hormones occur in enzymes might be generated from a higher concentrations than their biosingle precursor. With the determination logically active amidated counterparts 38. of the cDNA sequence of amidating Consequently, increasing the amidating enzymes ~.pti~ylglycine hydroxyl- activity in a cell might lead to an ases) from Xenopus~s.~9 and ox 3°, and increase in the amount of hormone
Peptide-COlSNH.CH2.CO2H
Peptide-COlSNH.CH.CO2H
I
OH
Peptide-COlSNH2 + CH.CO2H
II
0 Figure 1 Mechanism of enzyme-catalysedpeptide amidation1.
amide generated, and this could represent one level at which physiological regulation occurs. Regulation of amidation may take place at the gene level during post-translational processing, or by variation in the availability of cofactors. It is also possible that regulation might take place by control of access of the amidating enzyme to its substrate. Experiments have shown that amidating activity can be influenced by a variety of hormonal, drug and dietary factors 38. It has been reported 4° that depriving AtT-20 mouse pituitary tumour cells of copper by chronic treatment with disulphiram, a copper chelator, led to a reduction in the degree of amidation of the 'hinge' or 'joining' peptide, the peptide that links adrenocorticotropic hormone (ACTH) to the aminoterminal region of the ACTH-endorphin prohormone. Chronic treatment of rats
Ph.NH.N=CH.CO2H
Ph.NH.N=C.CO2H
I OH
Ph.NH.NH-C.CO2H
II 0
Rgure 2 Oxidation of glyoxylic acid phenylhydrazone to oxalic acid phenylhydrazidecatalysed by peptidyl glycine hydroxylase13. 113
TIBS 16-MARCH1991
preproenzyme residue number
0
100
200
300
400
I
I
I
I
I
500
600
I
I
700
I
800
900
1000
I
I
I
NH 2
I
Type A
NH 2
Type B
B/
I
c
c
I
Ii
.=
4
El il !!1
Active core
Rgure 3 Diagrammatic representation of amidating enzymes predicted by translation of cDNA sequences derived from a rat thyroid cDNA library by Bertelsen et al. 31 The signal sequences are indicated by the hatched areas, the amino terminus of the enzyme by NH2, putative transmembrane domains by the solid areas, potential glycosylation sites by C, and paired basic amino acid residues offering possible cleavage sites by the vertical dashed lines. The two predicted enzymes (Type A and Type B) appear to be formed by alternative gene splicing.
with disulphiram reduced the pituitary levels of both the 'hinge' peptide and MSH39. Similarly, depletion of ascorbate in cultured pars intermedia cells led to a reduction in the concentration of the amidated form of a-MSH relative to its glycine-extended precursoP L42.Anterior pituitary cells showed a similar effect: the amidating activity declined when ascorbate was removed from the culture media but partiaUy recovered when the levels were restored 4~. in the case of the guinea pig, which is unable to synthesize ascorbate, depletion in dietary levels of this potential cofactor can reduce the extent of amidation of pituitary (I-MSH43. However, it should be noted that the removal of copper or ascorbate may have wide ranging consequences apart from a possible influence on amidation, and it has not been established unequivocally that either copper or ascorbate is actually involved in the normal physiological regulation of amidation. Recently, 4-phenyi-3-butenoic acid was shown to be an in vivo inhibitor of peptidylglycine hydroxylase 44. It was found to bring about a reduction in the intracellular amidating activity in CA77 rat thyroid carcinoma cells and interestingly the results pointed to the existence of a homeostatic mechanism used by the cells for maintaining levels of amidating activity. The cells appear to respond to the presence of the inhibitor by producing more enzyme. In the same way, administration of diethyldithiocarbamate (DDC) to rats led to decreased production of gastrinamide and this was followed by an increase in ami114
surface protein. This approach might, for example, allow the amidation of gastrin to be controlled, which may be of value in the treatment of peptic ulcers. Furthermore, the application of an inhibitor could open the way to studying whether the enzyme fulfils a function in the cell in addition to peptide amidation. Since only two of the enzymes involved in mammalian prohormone processing have been positively identified, of which peptidylglycine hydroxylase is one, studies of the biosynthesis, intracellular transport and processing of this enzyme should greatly increase our understanding of how cells produce and control the activity of peptide hormones.
References 1 Bradbu~y,A. F., Rnnie, M. D. A. and Smyth, D. G. (1982) Nature 298, 686-688 dating enzyme synthesis 45. Also, glu- 2 Bradbury,A. F. and Smyth, D. G. (1987) Biosci. Rep. 7, 907-916 tathione, a tripeptide that is present in 3 Eipper, B. A. and Mains, R. E. (1988) Annu. most cells and possesses a carboxyRev. Physiol. 50, 333-344 terminal glycine, would be capable of 4 Meister, A. (1962) in The Enzymes (Vol. 6), pp. 443-445 competing in the amidation reaction by 5 Fruton, J. S., Johnston, R. B. and Fried, M. binding to the amidating enzyme. If this (1951) 1 Biol. Chem. 190, 39-53 were to occur, less enzyme would be 6 Bateman, R. C., Youngblood,W. W., Busby, available for the amidation of other W. H. and Kiser, J. S. (1985) J. BioL Chem. 260, 9088-9091 peptides. 7 Eipper, B. A., Mains, R. E. and Glembotski, C. C. (1983) Proc. Natl Acad. Sci. USA 80, Future directions 5144-5148 High-affinity antibodies have now 8 Landymore-Um,A., Bradbury, A. F. and Smyth, D, G. (1983) Biochem. Biophys. Res. been raised to synthetic peptides that Commun. 117, 289-293 correspond to regions of the sequence 9 Mizuno, K., Sakata, J., Kojima, M., Kangawa, K. of peptidylglyclne hydroxylase. This and Matsuo, H. (1986) Biochem. Biophys. Res. Commun. 137, 984-991 should allow immunocytochemical methods to be used for tracing the 10 Bradbury, A. F. and Smyth, D. G. (1985) in Biogenetics of Neurohormonal Peptides route of the enzyme from its biosyn(H~kanson, R. and Theorell, J., eds), thesis in the rough endoplasmic reticupp. 171-186, Academic Press lum to its appearance in the secretory 11 Markoslan, K, A., Paitian, N. A., Abramian, K. S. and Nalbandian, R. M. (1989) Biokhimicia 54, granule and ultimately to its secretion 2046-2053 from the cell surface. Since glycine- 12 Southan, C. and Kruse, L. I. (1989) FEBS Lett. extended precursors can occur in sig255, 116-120 nificant quantities 38, the question of 13 Bradbury, A. F. and Smyth, D. G. (1987) Eur. J. Biochem. 169, 579-584 where the substrate and amidating 14 Young, S. D. and Tamburini, P. P. (1989) J. Am. enzyme are brought together arises. Chem. Soc. 111, 1933-1934 Are they initially located in separate 15 Suzuki, J., Shimoi, H., Iwasaki, Y., Kawahara, T., Matsuura, Y. and Nishikawa, Y. (1990) EMBO J. compartments that undergo fusion 13, 4511-4517 before amidation takes place, or are the 16 Katopodis, A. G., Ping, D. and May, S. W. (1990) amidating enzyme and its substrates Biochemistry 29, 6116-6120 always present in the same compart- 17 Takahashi, K., Okamoto, H., Seino, H. and Noguchi, M. (1990) Biochem. Biophys. Res. ment, with the extent of amidation Commun. 169, 524-530 being a function of time and depending 18 Tajima, M., lida, T., Yoshida, S., Komatsu, K., on the availability of cofa(.~ors? Namba, R., Yanagi, M., Noguchi, M. and Okamoto, H. (1990) J. Biol. Chem. 265, The development of specific inhibitors of peptidylglycine hydroxyl- 19 9602-9605 Murthy, A. S. N., Mains, R. E. and Eipper, B. A. ase that penetrate cells raises the possi(1986) J. Biol. Chem. 261, 1815-1822 bility that the inhibitor might be targetted 20 Noguchi, M., Takahashi, K. and Okamoto, H. (1989) Arch. Biochem. Biophys. 275, to a tissue by attachment to an appro505-513 priate 'address' molecule, such as an 21 Eipper, B. A., Myers, A. C. and Mains, R. E. antibody with affinity for a specific cell(1985) Endocrinology 116, 2497-2504
TIBS16- MARCH1991 22 Mains, R. E. and Eippar, B, A. (1984) Endocrinology 115,1683-1690 23 Bradbury, A. F. and Smyth, D. G. (1983) Biochem. Biophys. Res. Commun. 112, 372-377 24 Tamburini, P. P., Jones, B. N., Consalvo, A.P., Young, S. D., Lovato, S. J., Gilligan, J.P., Wennogle, L. P., Erion, M. and Jeng, A. Y. (1988) Arch. Biochem. Biophys. 267, 623-631 25 Bradbury, A. E and Smyth, D. G. (1983) in Peptides, Structure and Function (Hruby, V. J. and Rich, D. H., eds), pp. 249-253, Pierce Chemical Company 26 Tamburini, R R, Young, S. D., Jones, B. N., Palmesino, R. A. and Consalvo, A. R (1990~ Int. J. Pept. Protein Res. 35,153-156 27 Suchanek, G. and Kreil, G. (1977) Proc~Natl Acad. Sci. USA 74, 975-978 28 Mizuno, K., Ohsuye, K., Wada, Y., Fuchimura, K., Tanaka, S. and Matsuo, H, (1987) Biochem. Biophys. Res. Commun. 148, 546-553 29 Ohsuye, K., Kitano, K., Wada, Y., Fuchimura, K.,
Tanaka, S., Mizuno, K. and Matsuo, H. (1968) Biochem. Biophys. Res. Commun. 150, 1275-1281 30 Eipper, B. A., Park, L. P., Dickerson, I. M., Keutmann, H. T., Thiele, E. A., Roddguez, H., Schofield, P. R. and Mains, R. E. ~1987) Mol. Endocrinol. 1, 777-790 31 Birtelsen, A. H., Beaudry,G. A., Galella, E. A., Jones, B. N., Ray,M, L. and Matea, N. M. (1990) Arch. Biochem. Biophys. 279, 87--96 32 Stoffers, D. A., Green, C. B-R. and Eipper, B. A. (1989) Proc. Natl Acad. Sci. USA 86, 735-739 33 Glauder, J., Ragg, H., Rauch, J. and Engels, J. W. (1990) Biochem. Biophys. Res. Commun. 169, 551-558 34 Murthy, A. S. N., Mains, R. E. and Eipper, B. A. (1986) J. BioL Chem. 261,1815-1822 35 Bradbury, A. F. and Smyth, D. G. (1988)in Progress in Endocrinology(Imura, H., Shizume, K. and Yoshida, S., eds), pp. 335-340, Elsevier Science Publishers 36 Bradbury, A. E and Smyth, D. G. (1988)
Biochem. Biophys. Res. Commun. 154, 1293-1300 37 May, V., Cullen, E. I., Braas, K. M. and Eipper, B. A. (1988) J, BioL Chem. 263, 7550-7554 38 Sugano, K., Park, J., Dobbins, W. D. and Yamada, J. (1987) Am. J. Physiol. 253, 6502-6507 39 Mains, R. E., Myers, A. C. and Eigper, B. A. (1985) Endocrinology 116, 2505-2515 40 Mains, R. E., Park, L. R and Eipper, B. A. (1986) J. Biol. Chem. 261,11938-11941 41 May, V. and Eipper, B. A. (1985) J. Biol. Chem. 260,16224-16231 42 Glembotski, C. C. (1986) Endocrinology 118, 1461-1468 43 Fenger, M. and Hilsted, L. (1988) Acta EndocrinoL 118, 119-125 44 Bradbury, A. F., Mistry, J., Roos, B. A. and Smyth, D. G. (1990) Eur. J. Biochem. 189, 363-368 45 Dickinson, C. J., Marino, L. and Yamada,T. (1990) Am. J. PhysioL 285, 6810-6814
REFLECTIONS onBIOCHEMISTRY DESPITE 135 RECENT downturn, the wool industry is of immense importance to the Australian economy, and Australians still talk of 'living off the sheep's back'. There are, in fact, roughly ten sheep for every person living in Australia. However, since World War II, wool has been steadily losing its market share to man-made fibres, until it now retains only 5% of the world textile market. Since the invention of nylon by Carothers in 1936, the threat of the man-made fibre industry to wool has been recognized by the Australian wool industry, so that in 1948 the Government appointed an expert committee to suggest ways to improve the utilization of wool. As a result of recommendations by this committee, the Wool Textile Research Laboratories were established within Australia's premier research body, the Commonwealth Scientific and Industrial Research Organization (CSIRO). One of these, the Parkville (Victoria) laboratory, was established to investigate the chemistry of the wool fibre. In 1959 this laboratory became the CSIRO Division of Protein Chemistry (DPC), which was responsible for much of the research on
Wool, a dead tissue of epithelial origin, derives many of its properties as a textile fibre from the structure and arrangement of the proteins from which it is comprised. Much of the progress in the elucidation of wool protein structures, as a step towards understanding this relationship between structure and properties, has been made in the Division of Protein Chemistry of Australia's Commonwealth Scientific and Industrial Research Organization.
the proteins of wool. The fundamental research on wool proteins at its zenith occupied some 13-14 of the 55 professional scientists on the staff at the DPC. Until the wool industry suffered a serious setback in the late 1960s and early 1970s, the DPC was almost entirely concerned with various aspects of wool chemistry, but after this time diversified its activities into other areas of biochemistry. The work of the DPC was at least partly responsible for Nobel Laureate Dr Arthur Kornberg stating in 1968, in a plea for support for biological research in the USA, ' . . . we know less about the molecul~s of our chromosomes and our brain than we do L. M. Dowllngand L. 6. Span~w are at CSIRO, about the molecules in Austra!i~n wool. Division of WoolTechnology,343 Royal This is because Australian wool growParade,Parkville,Victoria3052, Australia.
© 1991,ElsevierSciencePublishersLtd,(UK) 0376-5067/91/$02.00
ers have for many years recognized the value of basic research in the composition and chemical properties of wool.' It is an interesting coincidence that this basic wool research has revealed structures common to both wool and cells of the brain. The funds to support this research come partly from direct contributions from woolgrowers and are administered by the Wool Research Committee on which woolgrowers have a strong representation. Humans use wool for much the same purpose as does the sheep - for protection, warmth and decoration. These are all mechanical and structural uses rather than dynamic ones, so the prime study of wool is one of structure. Wool is a dead tissue of epithelial origin, and is classified as a hard a-keratin along
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