© 1987, Elsevier Science Publishers B.V (Biomedical Division) All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher, Elsevier Science Publishers B.V.(Biomedical Division), P.O Box 1527, 1000 BM Amsterdam (The Netherlands) No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein Because of the rapid advances in the medical sciences, the Publisher recommends that independent verification of diagnoses and drug dosages should be made Special regulation for readers in the U.S.A.: This publication has been registered with the Copyright Clearance Center Inc (Ccq, Salem, Massachusetts Information can be obtained from the CCC about conditions under which the photocopying of parts of this publication may be made in the U.S.A All other copyright questions, including photocopying outside of the U.S.A., should be referred to the publisher ISBN for the series: 0-444-80303-3 ISBN for the volume: 0-444-80886-8 Published by: Elsevier Science Publishers B.V (Biomedical Division) P.O Box 211 1000 AE Amsterdam (The Netherlands) Sole distributors for the U.S.A and Canada: Elsevier Science Publishing Company, Inc 52 Vanderbilt Avenue New York, NY 10017 (U.S.A.) Library of Congress Cataloging-in-Publication Data (Revised for volume 16) New comprehensive biochemistry Vol 16 published: Amsterdam; New York: Elsevier Science Publishers B.V (Biomedical Division); New York, N.Y U.S.A.: Sole distributors for the U.S.A and Canada, Elsevier Science Pub Co Includes bibliographical references and indexes Contents: v \ Membrane structure / editors, J.B Finean and R.H Michell v Stereochemistry / editor, Ch Tamm v Phospholipids / editors, J.N Hawthorne ana G.B Ansell [etc.] v 16 Hydrolytic enzymes / editors, A Neuberger and K Brocklehurst \ Biological chemistry Collected works I Neuberger, Albert II Deenen, Laurens L.M van [DNLM \ Membranes Anatomy and histology WI NE372F v.l / QS 532.5.M534] QD415.N48 574.19'2 81-3090 ISBN 0-444-80303-3 (Elsevier/North-Holland: set) Printed in The Netherlands Hydrolytic Enzymes Editors A NEUBERGER and K BROCKLEHURST The Lister Institute of Preventive Medicine, Charing Cross Hospital Medical School, St Dunstan's Rd., London W6 8RP(U.K.), and Department ofBiochemistry, Medical College of St Bartholomew's Hospital, University of London, Charterhouse Square, London ECl M 6BQ (U.K.) 1987 ELSEVIER AMSTERDAM· NEW YORK· OXFORD Preface A volume dedicated to Hydrolytic Enzymes was perceived as useful for two reasons In the first place, a number of these enzymes are not dealt with fully in those volumes of this series in which systems and events are discussed principally in a particular metabolic or physiological context Secondly, it seems appropriate to bring together discussion of some of the enzymes that became the focus of attention in the 1960s when our understanding of the function of protein molecules was revolutionised by the application of X-ray crystallography At that time, an account of the structure of myoglobin was rapidly followed by reports of the structures of lysozyme, carboxypeptidase, ribonuclease, chymotrypsin and papain, which permitted for the first time the results of mechanistic study by kinetic and protein chemical methods to be thought about within a realistic structural framework Six of the eight chapters are devoted to various aspects of proteolysis This emphasis is not inappropriate in view of the many advances in the search for chemical understanding of biological phenomena that were achieved during the study of proteolytic enzymes They were among the fIrst enzymes to be highly purified and crystallised and much of our understanding of molecular aspects of catalytic mechanism and specillcity is founded on the study of these enzymes Four of the chapters deal with the different types of proteinase that are differentiated on the basis of Hartley's idea of classifying proteinases by catalytic mechanism rather than by origin, specillcity or physiological function These chapters are complemented by one on proteinase inhibitors and by a short review of intracellular proteolysis The latter includes a brief discussion of ATP-dependent proteolysis by ubiquitin, which will be extended in a subsequent volume dealing with protein metabolism The final two chapters deal respectively with pancreatic ribonuclease A, the best characterised of the endoribonucleases, and with the phosphomonoesterases A particular regret, in view of the central importance oflysozyme in the development of studies on structure, specificity and mechanism, is the unavoidable omission of a chapter on glycosidases It is hoped that this omission will be rectified in a subsequent volume The major development of the 1960s in providing three-dimensional structures of enzymes at atomic resolution is being augmented in the 1980s by the application of DNA technology to provide designed structural variation in individual amino acid residues by site-directed mutagenesis This approach should go some way towards obviating the largest single problem that has held back mechanistic study of enzyme catalysis, namely the inability to vary systematically the structure of both or all of the reactant molecules We wish to record our thanks to the authors both for their excellent contributions and for their helpful cooperation in the editorial process London December 1987 A Neuberger K Brocklehurst A Neuberger and K Brocklehurst (Eds.), Hydrolytic Enzymes © 1987 Elsevier Science Publishers B.V (Biomedical Division) CHAPTER I Aspartyl proteinases JOSEPH s FRUTON Yale University New Haven CT (U.S.A.) Introduction (a) Historical background The aspartyl proteinases represent one of the four known main classes of enzymes that act at interior peptide bonds of proteins and oligopeptides (endopeptidases); the other classes are denoted serine proteinases, cysteine proteinases and metalloproteinases Because of their optimal action at pH 1.5-5, the aspartyl proteinases were previously named acid proteinases With the recognition that particular carboxyl groups in these enzymes are essential for catalysis, the term carboxyl proteinase was then used The identification of these groups as belonging to aspartyl residues in several members of this class has led to the currently-preferred terminology The term 'aspartyl proteinase' (aspartic proteinase and aspartate proteinase have also been used) is more appropriate than 'acid proteinase' because some enzymes now known to belong to this class act optimally on their substrates near pH Few enzymes occupy a more important place in the history of biochemistry than the one found in 1834 by Johann Nepomuk Eberle (1798-1834) in extracts of gastric mucosa Two years later, Theodor Schwann (1810-1882) characterized this 'ferment', named it pepsin, and established its physiological role in the mammalian digestion of food proteins [1] During the succeeding 60 years, pepsin was considered to be the prototype of the 'unorganized ferments' (KUhne named them enzymes in 1876) as distinct from the 'organized ferments' responsible for such processes as the fermentation of sugar by yeast [2] Many efforts were made to purify pepsin; the work of Ernst Wilhelm von Brncke (1819-1892) and of Comelis Adrianus Pekelharing (1848-1922) is especially noteworthy The high point came in 1930, when John Howard Northrop (b 1891) described the crystallization of pig pepsin [3] Although this achievement followed the crystallization of urease by James Sumner, it was Northrop's massive evidence for the protein nature of pepsin that led to the rejection of the view, advocated by Richard Willstatter during the 1920's, that enzymes are small catalytic molecules adsorbed on inactive protein carriers [4] Another important discovery made in this field before 1900 was the observation by John Newport Langley (1852-1925) that a slightly alkaline extract of gastric mucosa contains a material (pepsinogen) which is converted to pepsin on acidification of the extract [5] The crystallization of pig pepsinogen in 1938 by Roger Moss Herriott (b 1908) made possible the incisive study of its conversion to pepsin [6] The work of Northrop and Herriott thus marks the beginning of the modem study of pepsin as a protein and as a catalytic agent, and has influenced the investigation of the other enzymes now considered to be aspartyl proteinases (b) Occurrence and nomenclature In all vertebrates the gastric juice contains one or more pepsins that arise from secreted pepsinogens; the latter are produced mainly in the chief cells (zymogen cells) of the fundus (corpus) [7,8] The secretion of the pepsinogens is under vagal control both directly on the oxyntic glands of the fundus and indirectly through the release of peptide hormones (gastrins) from the pyloric glands [9] Multiple forms of pepsinogen, and of the pepsins derived from them have been found in many vertebrates (e.g., man, monkey, pig, beef, rat, chicken, dogfish) Chromatographic separation of the components has shown that the predominant pepsin A (usually denoted pepsin) of adult mammals is accompanied by pepsin C (the currentlypreferred name is gastricsin), as well as by the minor components denoted pepsin B and pepsin D [10] Some investigators have given the individual chromatographic components Roman numerals, while others have numbered the gastric proteinases and their zymogens in the order of decreasing electrophoretic mobility at pH 5.0 or 8.5 respectively [11] Immunochemical methods have also been applied to the differentiation and numbering of human gastric proteinases [12] In the gastric juice offetal and newborn mammals the major pepsin-like enzyme is chymosin (derived from the zymogen prochymosin); this was its original name [13], but for many years it was called rennin because it is the chief enzymic component of rennet, the calf-stomach (abomasus) extract used in the manufacture of cheese [14] Regrettably, the nomenclature of the mammalian gastric proteinases has been in a confused state because of differences in the terminology used by various groups of investigators; for a helpful clarification, see Foltmann [15] Among the aspartyl proteinases from vertebrates is the kidney enzyme renin (also present in submaxillary tissue), whose important physiological function is the formation from plasma angiotensinogen of the decapeptide angiotensin I, which is in tum cleaved by a 'converting enzyme' to the highly active pressor octapeptide angiotensin II [ 16,17] It should be noted that although renin is now known to be an aspartyl proteinase, it is not an acid proteinase, since the pH optimum for its action is 6-8 Another aspartyl proteinase is the lysosomal enzyme cathepsin D present in many animal tissues (spleen [18], liver [19], uterus [20,21], thyroid [22], skeletal muscle [23], anterior pituitary [24], brain [25], seminal tissue [26], erythrocytes [27], lymphoid tissue [28]) A cathepsin D and its inactive precursor from monkey lung appears to resemble gastricsin and its zymogen [29] Among the aspartyl proteinases in plants are those present in Lotus seed [30] and in the insectivorous plants Nepenthes and Drosera [31) There has been considerable interest in microbial acid proteinases in part because of a search for suitable rennet substrates The enzymes subjected to the most intensive study have been penicillopepsin (from Penicillium janthinellum) [32], Rhizopus-pepsin (from Rhizopus chinensis) [33], and the acid proteinases from Endothia parasitica [34] and Mucor miehei [35] Well-characterized microbial acid proteinases have also been isolated, and in some cases crystallized, from strains of Acrocylindricum sp [36], Aspergillus saitoi [37], Candida albicans [38], Cladosporium sp [39], Fusarium moniliforme [40], Monascus kaoling [41], Mucor pusillus [42], Paecilomyces varioti [43], Penicillium duponti [44], Rhodotorula glutinis [45], Russula decolorans [46], Tramestes sanguinea [47], Trichoderma viride [48], and both Saccharomyces cerevisiae and S carlsbergensis (yeast proteinase A) [49] No evidence is available for the existence of zymogens for the above microbial proteinases The enzymes from Endothia parasitica, Mucor miehei and Mucor pusillus have been used in cheese manufacture These various aspartyl proteinases have in common the property of cleaving proteins (e.g., denatured hemoglobin, serum albumin) and suitable oligopeptides at pH 1.5-5.5 A widely-used diagnostic test is their inhibition by the naturally-occurring peptide pepstatin and by active-site-directed diazo compounds; these properties will be discussed later in this chapter Although future study of other enzymes may show them to belong to the aspartyl proteinase family, in what follows primary attention will be given to those known to exhibit these properties Among the acid proteinases that not appear to be inhibited by pepstatin or diazo compounds is the one from Scytalidium Iignicolumn [50] Apart from the confusion in the naming of the mammalian gastric proteinases, mentioned previously, the nomenclature of the acid proteinases has not been aided by the Commission on Enzymes of the International Union of Biochemistry [51] Proteolytic enzymes belonging to different classes have been given the same name and distinguished from each other only by the addition of a different capital letter and the assignment of different numbers Moreover, the Commission has retained the longoutworn distinction between hydrolases and transferases for enzymes that act on peptide, ester and glycosidic bonds presented in the first edition of the treatise of Dixon and Webb [52] (c) Purification In his work on crystalline pig pepsin, Northrop noted that the preparations differed considerably in homogeneity, as indicated by measurement of their solubility behavior Part of the inhomogeneity was a consequence of the presence of peptide material, formed by autodigestion Subsequently, Steinhardt performed a careful study of the solubility properties of crystalline pig pepsin, and gave clear evidence of its inhomogeneity as a protein [53] After the introduction of ion-exchange chromatography for the fractionation of proteins, Ryle and his associates established the presence of the minor components mentioned previously [10] Additional factors that may contribute to heterogeneity are the presence of multiple gene products (made evident by amino acid substitutions) and different degrees of phosphorylation [54] or glycosylation [55] At present, the preferred method for the preparation of apparently homogeneous pig pepsin A is rapid activation of crystalline pepsinogen (shown to be homogeneous by several criteria [56]), and passage of the mixture first through sulfoethyl Sephadex C-25 to remove peptides and then through Sephadex G-25 to remove salts This pepsin preparation is homogeneous on hydroxylapatite or DEAE-cellulose, which may also be used to effect the purification of commercial preparations of crystalline pig pepsin [57] Similar chromatographic procedures have been used for other aspartyl proteinases Some aspartyl proteinases have been purified by means of affmity chromatography on columns of Sepharose 4B or agarose to which a substrate analogue (e.g., L-Phe-D-Phe) [58] or pepstatin [59] has been attached by means of aminohexoyl bridges; hemoglobin-agarose columns have also been employed [60] The purification of cathepsin from various animal tissues has been attended with difficulty, as is indicated by the heterogeneity of the enzyme preparations that have been described [61-64] Crystallization has been usually effected by means of ammonium sulfate or acetone Aside from pig pepsin A, the following aspartyl proteinases have been obtained in crystalline form: the pepsins from beef [65] and salmon [66], calf chymosin [67], penicillopepsin [32], Rhizopus-pepsin [33], and the proteinases from Endothia parasitica [34], Aspergillus saitoi [37], Mucor pusi//us [42], Paeci/omycetes varioti [43], Penicillium duponti [44], Rhodotoru/a glutinis [45], and Trametes sanguinea [47] The crystallization of chicken liver cathepsin has been reported [68] (d) Assay A widely-used method is that introduced by Anson [69]; later investigators have made slight modifications in the procedure Acid-denatured hemoglobin is the substrate at pH 1.8 and 37°C, and the release of cleavage products that are soluble in 3% trichloroacetic acid is measured spectrophotometrically at 280 nm One unit of pepsin activity is usually defmed as the amount of enzyme that produces an increase in absorbance of 0.001 per minute under the conditions of the assay Commercial preparations of crystalline pig pepsin generally contain 2500-3000 units per milligram; the material obtained by chromatographic purification of pepsin produced by the rapid activation of pepsinogen assays at about 4000 units per milligram [57] Other assay methods with protein substrates include the use of bovine serum albumin in place of hemoglobin For chymosin and related acid proteinases, a useful assay procedure is the measurement of the rate of clotting of 10% reconstituted skim milk powder in the presence of CaCl [14] Some microbial acid proteinases catalyze the activation of trypsinogen at pH 3.4; the assay method introduced by Kunitz [70] has been modified [32] Various synthetic peptides have been used in the assay of the aspartyl proteinases Among these substrates are compounds of the type A-Phe(N0 2)-Y-B· (where Y = Phe, Leu etc.); the rate of cleavage of the Phe(N02)-Y bond may be followed spectrophotometrically at 310 nm [71] Older methods have involved the use of substrates of the type A-Phe-Y-OH (where Y = Tyr(I2)' Phe etc.), and measurement of the rate of hydrolysis of the Phe-Y bond by the ninhydrin method [72]; this procedure has been automated [73] Molecular properties (a) Physical-chemical properties By means of the sedimentation-equilibrium method, values of 32700 ± 1200 and 40400 ± 1600 were obtained for the molecular weight of pig pepsin A and pepsinogen A respectively [74] These values may be compared to 34644 and 39637 calculated from the amino acid sequences Other methods (for example, sedimentation-velocitydiffusion, light scattering, osmotic pressure) gave values for pepsin ranging from 32000 to 35000 Estimates of the molecular weight of other gastric proteinases and ofmicrobial acid proteinases (in some cases determined by means of sodium dodecyl sulfate-agar gel electrophoresis) have given values ranging from about 31 000 to about 40000; for the zymogens the values range between 36000 and 43000 [75] The aspartyl proteinases are acidic proteins, as a consequence of the preponderance of dicarboxylic acid residues as compared to the basic amino acid residues In the case of pig pepsin, the paucity of lysine and arginine residues is especially marked Early studies by Tiselius and Herriott suggested that the isoe1ectric point of pig pepsin lies below 1, since the protein still migrated as an anion at this pH value This conclusion is probably incorrect, as is suggested by more recent studies in which the isoelectric focussing technique was employed [76-78] However, in view of the heterogeneity of the preparations, and the extended time required in this method, no defmite isoelectric point can be assigned, except to infer that pepsin A has a pI between and In the case of other aspartyl proteinases where the balance between acidic and basic amino acids is less extreme, the isoelectric points are between and In contrast to the extremely low isoelectric point of pig pepsin, that of pig pepsinogen is about 3.7; this difference is consistent with the cationic character of the peptide removed from the zymogen upon its conversion to pepsin (see Section 3(a)) • Abbreviations (in alphabetical order) used in this chapter and not defined in the Recommendations of the IUPAC-IUB Joint Commission on Nomenclature on the Nomenclature and Symbolism for Amino Acids and Peptides [Eur J Biochem (1984) 138,9-37]: DAN, diazoacetyl-nt-norleucine methyl ester; Dns, 5-dimethylamino-I-naphthalenesulfonyl; EPNP, 1,2-epoxy-3-(p-nitrophenoxy)propane; Mns, 6-(Nmethylanilino)-2-naphthalenesulfonyl; Nle, L-norleucyl; Nva, L-norvalyl; OP4P, 3-(4-pyridyl)propyl-I-oxy; Phe(N0 ) , p-nitro-L-phenylalanyl; Pia, p-phenyl-L-Iactyl;Pol, L-phenylalaninol; TPDM, p-toluenesulfonylt-phenylalanyldiazomethane; TNS, 2-p-toluidinylnaphthalene-6-sulfonate; Tyr(I ) , 3,5-diiodo-L-tyrosyl The abbreviated designation of amino acid residues denotes the L form, except where otherwise indicated Highly-purified preparations of several aspartyl proteinases exhibit the presence of multiple components on isoelectric focussing Among them are beef spleen cathepsin D [61], and Rhizopus pepsin [79] (b) Amino acid composition and sequence Many investigators have studied the amino acid sequence of segments of pig pepsin A; the complete sequence proposed by Sepuvelda et al [80], has been widely accepted (Fig 1) The amino acid composition implied by this sequence (total, 327 residues) differs from that reported (total, 321 residues) by Rajagopalan, Stein and Moore [57] There are several notable features in the sequence: (1) the paucity of strongly basic side-chain cationic groups (l Lys, Arg), all located within the 20-amino acid carboxylterminal section of the protein; (2) the overwhelming predominance of side-chain carboxyl groups; (3) the presence of a relatively large number of residues of hydroxyamino acids, of proline and of aromatic amino acids; (4) the presence of three disulfide bridges in relatively small loops Earlier work had shown that pig pepsin A contains one phosphoryl group per molecule [81], and that this group is absent in both pig pepsin D and its zymogen [82]; in the sequence shown it is Ser-68 that is phosphorylated The sequence presented in Fig is for the major component of the enzyme preparation analyzed by Sepuvelda et al [80] Some of the pepsin molecules had an additional H-Ala-Leu- unit at the amino terminus or a deletion of Ile-230 or the replacement of Ser-255 by a glutamine residue In the conversion of pig pepsinogen A to pepsin A, the 44-residue amino-terminal action of the zymogen is removed The amino acid sequence of this fragment has been reported [83] to be: H-Leu-Val-Lys-Val-Pro-Leu-Val-Arg-Lys-Lys-Ser-Leu-Arg-Gln-Asn-Leu-Ile-Lys-Asp-Gly-Lys-Leu-Lys-Asp-Phe-Leu-Lys-Thr-His-Lys-His-Asn- Pro- Ala-Ser-Lys-Tyr-Phe- Pro-Glu-Ala-Ala-Ala-LeuIt is evident that this peptide contains most of the strongly basic residues of pig pepsinogen A, thus accounting for the marked difference in the isoelectric points of the zymogen and the active enzyme The amino acid composition of pepsinogen A and pepsin A from other mammalian species (man, monkey, beef, rat) resembles those from the pig, and extensive sequence homology is evident [84,85] In all cases studied, the gastric proteinases have halfcystine residues to form three disulfide bridges In chicken pepsin (and pepsinogen), however, in addition to these bridges there is a cysteinyl residue [55] It is also noteworthy that chicken pepsin has a larger proportion of strongly basic amino acids (8 Lys + Arg) and an isoionic point near pH 4; the small net negative charge of this enzyme may account for its stability at pH values above 6, where pig pepsin is rapidly denatured [86] A complete amino acid sequence has been reported for calf chymosin, and its zymogen prochymosin [87] The latter resembles pig pepsinogen A in having 365 amino 10 20 30 40 H-l1e-Gly-Asp-Glu-Pro-Leu-Glu-Asn-Tyr-Leu-Asp-Thr-Glu-Tyr-Phe-Gly-Thr-l1e-Gly-IleGly-Thr-Pro-Ala-Gln-Asp-Phe-Thr-Val-Ile-Phe-Asp-Thr-Gly-Ser-Ser-Asn-Leu-Trp-Val- I I 51 60 Pro-Ser-Val-Tyr-Cys-Ser-Ser-Leu-Ala-Cys-Ser-Asp-His-Asn-Gln-Phe-Asn-Pro-Asp-Asp70 80 90 100 110 120 130 140 150 160 170 180 Ser-Ser-Thr-Phe-Glu-Ala-Thr-Ser-Gln-Glu-Leu-Ser-Ile-Thr-Tyr-Gly-Thr-Gly-Ser-MetThr-Gly-Ile-Leu-Gly-Tyr-Asp-Thr-Val-Gln-Val-Gly-Gly-Ile-Ser-Asp-Thr-Asn-Gln-IlePhe-Gly-Leu-Ser-Glu-Thr-Glu-Pro-Gly-Ser-Phe-Leu-Tyr-Tyr-Ala-Pro-Phe-Asp-Gly-IleLeu-Gly-Leu-Ala-Tyr-Pro-Ser-Ile-Ser-Ala-Ser-Gly-Ala-Thr-Pro-Val-Phe-Asp-Asn-LeuTrp-Asp-Gln-Gly-Leu-Val-Ser-Gln-Asp-Leu-Phe-Ser-Val-Tyr-Leu-Ser-Ser-Asn-Asp-AspSer-Gly-Ser-Val-Val-Leu-Leu-Gly-Gly-Ile-Asp-Ser-S~r-Tyr-Tyr-Thr-Gly-Ser-Leu-Asn- 190 200 Trp-Val-Pro-Val-Ser-Val-Glu-Gly-Tyr-Trp-Gln-l1e-Thr-Leu-Asp-Ser-Ile-Thr-Met-Asp- I I 211 220 Gly-Glu-Thr-Ile-Ala-Cys-Ser-Gly-Gly-Cys-Gln-Ala-Ile-Val-Asp-Thr-Gly-Thr-Ser-Leu230 240 Leu-Thr-Gly-Pro-Thr-Ser-Ala-Ile-Ala-Ile-Asn-Ile-Gln-Ser-Asp-Ile-Gly-Ala-Ser-Glu251 260 Asn-Ser-A5p-Gly-Glu-Met-Val-Ile-Ser-Cys-Ser-Ser-Ile-Asp-Ser-Leu-Pro-Asp-Ile-Val270 280 290 300 310 320 Phe-Thr-Ile-Asp-Gly-Val-Gln-Tyr-Pro-Leu-Ser-Pro-Ser-Ala-Tyr-Ile-Leu-Gln-Asp-AspAsp-Ser-Cys-Thr-Ser-Gly-Phe-Glu-Gly-Met-Asp-Val-Pro-Thr-Ser-Scr-Gly-Glu-Leu-TrpIle-Leu-Gly-Asp-Val-Phe-Ile-Arg-Gln-Tyr-Tyr-Thr-Val-Phe-Asp-Arg-Ala-Asn-Asn-Lys327 Val-Gly-Leu-Ala-Pro-Val-Ala-OH Fig I Amino acid sequence of pig pepsin A (from ref 80) acid residues, and 42 residues are removed from the amino terminus upon activation to chymosin Like other mammalian gastric proteinases, chymosin has disulfide bridges, but in contrast to pig pepsin A (and like chicken pepsin) chymosin has a relatively large proportion of strongly basic amino acids (9 Lys + Arg) In contrast to the aspartyl proteinases from the mammalian gastric mucosa, which are single-polypeptide-chain proteins, the enzyme renin (from mouse submaxillary glands) has two chains linked by one disulfide bridge [88] Similarly, cathepsin D (from 409 amino acid sequence, 291-292 homologues, 292-293 kinetic properties, 291 physical properties, 291 specificity, 291 stability, 291 synthesis, 294 three-dimensional structure, 291-292 Bowman-Birk Proteinase Inhibitor (BBI) family, 273-282,287 active fragments from, 275-276 activity against trypsin and chymotrypsin, 272 amino acid sequences, 282-283 anticarcinogen activity, 278 chickpea inhibitor (CI) as analogue of, 281 Cicer arietinum inhibitor as analogue of, 281 covalent structure, 274 crystallographic study, 277 dietary, 278 dual independent activity, 273 evolutionary aspects, 278 garden bean trypsin inhibitors (GBI) as analogues of, 281-282 groundnut inhibitor (GI) as analogue of, 219-281 inhibitors analogous to, 278-282 isoinhibitors, 278 kinetics, 273-274 Lima bean inhibitor (LBI) as analogue of, 278-279 modification, 275-276 molecular weight, 214-275 nutritional significance, 277 pancreatic hypertrophy by, 277-218 peanut inhibitor as analogue of, 279-281 Phaseolus vulgaris trypsin inhibitors as analogues of, 281-282 physiological significance, 271-278 reactive site model and structure, 214 replacements in, 275-216 scission of,275-216 self-association, 274-215 specificity, 273-274 spectra, 275 stability, 273-274 structure, 274 synthesis, 276-271 therapeutic applications, 278 X-ray crystallographic study, 271 X-ray induced transformation: suppression by, 218 Bromelain (see stem bromelain and fruit bromelain) C.-inactivator, 262 Cl-inhibitor, 262 Cadmium in alkaline phosphatase, 390, 392 Calcium ions in regulation of intracellular proteolysis, 315, 319, 321 Calotropin DI catalytic properties, 82-83 folding pattern, 82 isoelectric point, 43 purification, 64 structural data, 40-42, 82-83, 126 three-dimensional structure, 82-83 X-ray diffraction studies, 40, 82-83 Calotropin FI, 42 Calpain(s) assay of catalytic activity, 74 characteristics, 57-58 discovery, 56-57 Gly in catalytic site region, 40 immunological studies, 79-80 inhibition by calpastatin, 60 isoeleetric point, 42 physiological and pathological aspects, 60-62 purification, 66 regulation of activity, 60-62 specificity, 100 structural data, 41-42 substrates for, 58, 14 Carbamylphosphate synthase (half-life 01), 308 Carboxypeptidase A, 201-255 acyl intermediate? in, 235-237 affinity chromatography for removal of ATEEase, 202, 219 anion binding, 224 anomalies in kinetic behaviour, 208 Arg-145 in, 202, 220-221 arginine modification in, 220-221 arginyl residues as specificity sites, 220-221 ATEEase as contaminant, 202 azo-Tyr-248-, 217 binding site residues, 202, 214-221 CABS-Sepharose as affinity gel for removal of ATEEase,202,219 carboxylic inhibitors, 211-213, 224 catalytic site residues, 202, 214-221 Cd(I1)-, 222-223 410 chemical modification, 214-221 chemical nature of ES intermediates, 241-243 chromophoric metal atoms in, 233 cis peptide bonds in, 204-205 Co(1l)- (see also Cobalt carboxypeptidase A), 227-232 Co(I1I)-, 227 cobalt-substituted,222 comparison of Zn(lI) and Cd(II) forms, 222-223 competitive inhibition, 211-213 conformational forms of ligand complexes, 217 coordination of zinc atom in, 206 cross-linked, 206 cryokinetics, 233-234 cryoquench studies, 241 cryospectrokinetic studies, 219-220,232 cryospectroscopy, 219-230,232,237-241 depsipeptides as substrates for, 207, 233, 241, 243 electrophilic catalysis, 222 enhancement of ligand nucleophilicity in, 222 EPR spectra 231, 240-241, 244 ES intermediates from peptides and from esters, 237-246 ES2 peptide intermediate 245 essential amino acid residues, 202, 214-221, 225 function of the metal, 222-227 functional tyrosyl residue, 216-220 general base catalysis 249 Glu-270 in 202, 211 214-215.225 glutamic acid modification, 214-215 Gly-Tyr complex, 206 231 hydrolysis of peptide substrates, 246-249 inactivity of Co(III}, 227 inhibitor interactions, 206-207 inhibitors, 211-213,242 intermediates (see Carboxypeptidase A Intermediates) intermolecular contacts in crystal structure, 205-206 isomerization of EH forms 213 kinetic behaviour, 208 «; time domain, 248 Lewis acid catalysis by, 222, 227 ligand complexes, 217 metal's function, 222-227 metal-bound H molecule, 211, 225 metal-bound intermediate 249 metal-hydroxide mechanism, 227 N-dansylated peptides as substrates for, 233 nitration of, 218-220 nitro-, 219 N0 2-Tyr-248-, 219 non-competitive inhibition, 211-213, 242 nueleophilicity enhancement in, 222 ortho nitration of, 218-220 peptide intermediate spectra, 244 peptide substrate hydrolysis, 246-249 peptide substrates for, 207 pH-rate profiles, 209-211 pKa values influencing kinetic behaviour of, 211 pre-steady state kinetics, 233-235, 248 primary structure, 202-204 radiationless energy transfer (RET) studies, 232.234 rate-determining step in catalysis of depsipeptide hydrolysis, 243 rate-determining step in catalysis of ester and peptide hydrolysis, 248-249 reactions catalysed by, 207-209 'reverse' protonation scheme 211 reversibility of reaction catalysed by, 243 site-directed mutagenesis, 221-222 spectrokinetic studies of nitro-, 219-220 spectroscopic studies (see Cobalt carboxypeptidase A) steady state kinetics, 233 structure-function relationships, 233 substrates for, 207-208, 233, 246-249 subzero temperature studies, 232, 234-235 temperature-jump studies, 213 template for alignment of reactants, 222 ternary enzyme peptide inhibitor (lES) complex, 242 tertiary structure, 204-207 thioamide substrates for, 208 three-protouation-state model, 209, 212-213 transient metal complexes 241 Tyr-248 in, 202.211, 216-220 tyrosine modification, 216-220 X-ray diffraction analysis, 204-207 zinc atom in (see also Zinc atom in Carboxypeptidase A), 206 222-227 Zn(I1}, 222-223 Carboxypeptidase A Intermediates, 233-249 acyl-, 235-237 ES 2- , 237-249 estero, 237, 248-249 411 rnetallo-, 241 247, 249 peptide-, 237, 242, 244-245, 248-249 Catalase (half-life of), 308 Cathepsin B (see also thiol-dependent cathepsins) active site titration, 75-77, 110 acyl enzyme mechanism, 131 acylation step of catalytic act, 137 amino acid sequence, 91-94, 126 assay of catalytic activity, 13-14 benzofuroxan as chromophoric oxidizing agent and reactivity probe for, 113, 131 carbohydrate attachments, 93 catalytic site characteristics, 56 CD spectra, 119 eDNA clone, 56 conformer B geometry of dithioester derivatives, 121 dipeptidylcarboxypeptidase activity, 101 folding pattern, 93 Gly in catalytic site region, 40, 126 high M, forms, 44,59,80 immunological studies, 19-80 interacti ve system in catalytic site, 109-110 ion-pair, 109-110, 137 isoelectric point, 42 mechanism, 140 nucleophilic competition studies, 137 pH-dependence of kco./Km, 131 physiological and pathological aspects, 58-60 2-pyridyl disulphide probes for, 110 purification, 65 reactivity probe s for, II 0, 113 rR spectroscopic studies, 121 sources, 55 specificity, 99-10I structural data, 41-42, 91-94, 126 subsite structure, 99-100 substrates for, 73-74 zymogens, 43-44 Cathepsin D, amino acid sequence, 1-8 angiotensin I release by, 22 p-endorphin cleavage by, 17 y-endorphin formation by action of, 11 insulin as subs trate for, 18 p-Iipotronin cleavage by, 11 primary specificity, 22 purification, secondary interactions with substrates, 26 Cathepsin H (see also thiol-dependent cathepsins) active site titration, 16 amino acid sequence, 91-93 Asn in catalytic site region, 40 ass ay of catalytic activity, 14 carbohydrate attachments, 93 folding pattern, 93 immunological studies, 19-80 interactive system in catalytic site, 109-110 ion pair, 109-110 isoelectric point, 43 mechanism, 140 physiological and pathological aspects, 58-59 2-pyridyl disulphide probes for, 110 purification, 66 reactivity probes for, 110 specificity, 99-I0 I structural data, 41, 91-93 substrates for, 74 Cathepsin L (see also thiol-dependent cathepsins] assay of catalytic activity, 14 immunological studies, 80 isoelectric point, 43 physiological and pathological aspects, 58-59 purification, 66 specificity, 100-101 structural data, 41 substrates for, 14 Cathepsin N (see also thiol-dependent carhepsins) assay of catalytic activity, 74 physiological and pathological aspects, 59 purification, 66 substrates for 74 Cellular autofiltration in regulation of intracellular proteolysis, 322 lIlCd-NMR spectra of alkaline phosphatase, 385-387,390 Chemical modification - basis of the technique and application [0 cysteine protein ases, 101-116 Chymopapain A active site titration, 76 imidazole photo-oxidaticn of, 115 isoelectric point, 43 photo-oxid ation of, 115 purification, 64,69-70 2-pyridyl disulphide probes for, lOS-III reactivity probes for, 105-114 specificity, 99 structural data, 41-42 Chymopapain B 412 active site titration, 76 as Cys-22 > Ser cysteine proteinase variant, 127 as Gln-19 > Val cysteine proteinase variant, 127 catalytic site, 94 interactive system in catalytic site, 109-110 ion pair, 109-110 isoelectric point, 43 purification, 69-70 2-pyridyl disulphide probes for, lOS-III reactivity probes for, 105-114 structural data, 41-42 Chymopapain(s) (see also Chymopapains A and B) as contaminants of papain, 69 conformer B geometry of dithioester derivatives, 121 discovery, 5I fractionation, 51-53 FTIR spectroscopic studies, 119 identilication, I-53 immunological studies, 80 purification, 69-70 2-pyridyl disulphide probes for, 105-111 reactivity probes for, 105-114 rR spectroscopic studies, 121 specificity, 99 thionoester substrates for, 133 Chymosin2 amino acid sequence, 6-7 assay, casein cleavage by, 17 insulin as substrate for, 18 kappa-casein cleavage by, 17 milk-clotting activity, 17 pepstatin as inhibitor of, 12 primary specificity, 21-22 reference substrate for, 21 three-di rnension aI crystal structure, 14-15 Chymotrypsin acetyl derivative, 163 acyl-enzyme intermediate formation, 162-165 acylation step of catalysis, 166-168 aminolysis of acyl enzymes, 179-180 catalytic site structure, I71-1 72 catalytic triad, 169-172 cinnamoyl imidazole as non-specific substrate, 163 deacylation of acyl enzymes by amines, 179-180 deaeylation step of catalysis, 166-168, 189 diisopropylphosphofluoridate as inhibitor, 162, 180 general base catalysis, 165 kinetic isotope effects, 166, I89 kinetic studies with added n ucleophiles, 164 kinetic studies with p-nitrophenylacetate, 162-163 NMR studies on transition state analogues, 189 oxyanion binding site, 169-172 peptide synthes is catalysis by, 179 pH-dependent kinetic studies, 165-169 phenylmethanesulfonylfluoride as inhibitor, 162, ISO primary specificity, 161, 172-173 proton inventory studies, 189 reviews, 159 secondary specificity, 174-176 specificity, 161, 172-176 stereochemistry of catalysis, 169-170 structural relationship to other serine proteases, 160 tetrahedral intermediates, 168-169, 189 thiono substrates for, 186 three dimensional structure, 171-1 73 X-ray diffraction studies, 171-173 Chymotrypsinogen activation of, 161 Clostripain assay of catalytic activity, 73 discovery, 54 isoelectric point, 42 kinetic studies, 137 pH-dependence of k