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PubMed1993

Heterogeneity of mast cells at multiple body sites. Fluorescent determination of avidin binding and immunofluorescent determination of chymase, tryptase, and carboxypeptidase content.

Human mast cells (MCs) from multiple sites were studied to determine heterogeneity of expression of chymase, tryptase, and/or carboxypeptidase and the binding to avidin. Three immunophenotypes were found: MCs positive for tryptase (MCT), no immunodetectable chymase; MCs positive for tryptase and chymase (MCTC); and MCs positive for chymase (MCC), no immunodetectable tryptase. Chymase-positive MCs also bound avidin and contained carboxypeptidase. In breast skin and parenchyma and axillary lymph nodes > 95% were MCTC; a rare MC in skin and lymph nodes was MCT or MCC. In lung alveoli 91% of MCs were MCT, 8% were MCTC and 1% MCC. In bowel mucosa 58% of MCs were MCT, 35% were MCTC and 7% MCC, whereas in bowel submucosa 83% of MCs were MCTC and 17% MCC. Within esophageal epithelium 38% were MCT and 62% MCTC; whereas, in esophageal subepithelium all were MCTC. This study further documents site-dependent diversity among normal human MCs and recognizes a novel third immunophenotype rich in chymase and relatively deficient in tryptase, the MCC cell.

Avidin↗

PubMed1997

Rapid induction by a blood meal of a carboxypeptidase gene in the gut of the mosquito Anopheles gambiae.

A search for genes induced rapidly (< 3 h) after a blood meal in the gut of the human malaria vector Anopheles gambiae led to the identification of a carboxypeptidase gene (AgCP). We report the sequence of the 1302 nt AgCP transcribed sequence, 710 nt of upstream and 585 nt of downstream DNA. The AgCP open reading frame is 60.4% identical at the nucleotide level to a blackfly, Simulium vittatum, carboxypeptidase gene. The transcriptional start site of AgCP was determined by primer extension. Expression of AgCP mRNA is detectable in the guts of pupae and sugar-fed adult female mosquitoes and is induced (approximately 10-fold) within 3 h of a blood meal. By 24 h after a blood meal, mRNA abundance returns to a level close to that present before a blood meal. Whole-mount in situ hybridization shows that AgCP mRNA expression is restricted to most or all cells of the posterior midgut. Expression of the AgCP and trypsin genes were compared and shown to differ in two fundamental ways: (1) the peak of AgCP expression after a blood meal occurs approximately 20 h before that of trypsin; and (2) induction of the AgCP gene is independent of the composition of the ingested meal whereas trypsin induction requires the presence of protein. The potential use of the AgCP promoter for driving the expression of genes that hinder the development of parasites in the mosquito gut is discussed.

Amino Acid Sequence↗

PubMed2002

Crystal structure of a novel carboxypeptidase from the hyperthermophilic archaeon Pyrococcus furiosus.

The structure of Pyrococcus furiosus carboxypeptidase (PfuCP) has been determined to 2.2 A resolution using multiwavelength anomalous diffraction (MAD) methods. PfuCP represents the first structure of the new M32 family of carboxypeptidases. The overall structure is comprised of a homodimer. Each subunit is mostly helical with its most pronounced feature being a deep substrate binding groove. The active site lies at the bottom of this groove and contains an HEXXH motif that coordinates the metal ion required for catalysis. Surprisingly, the structure is similar to the recently reported rat neurolysin. Comparison of these structures as well as sequence analyses with other homologous proteins reveal several conserved residues. The roles for these conserved residues in the catalytic mechanism are inferred based on modeling and their location.

Amino Acid Sequence↗

PubMed1997

C-terminal sequence analysis of peptides and proteins using carboxypeptidases and mass spectrometry after derivatization of Lys and Cys residues.

C-Terminal sequence analysis of peptides and proteins using carboxypeptidase digestion in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is convenient for protein and peptide characterization. After a short digestion, a sequence up to 20 residues can be identified, but the total number depends on the individual sequence. Due to the accuracy limits of the MALDI time-of-flight arrangement, the assignment of several residues with close mass values, including Lys/Glx, may remain ambiguous. We have used derivatization of lysine residues by guanidination to overcome the problem of Lys identification. The reaction is rapid and specific and results in full derivatization. In the case of Cys-containing peptides, problems arise from the fact that carboxypeptidases Y and P do not cleave peptides that contain nonderivatized cystine, cysteic acid, or (carboxymethyl)cysteine. Successful identification of Cys residues within the sequence is instead achieved by conversion of Cys to 4-thialaminine by (trimethylamino)-ethylation. The two derivatizations of Lys and Cys side chains provide opportunities for proton attachment and therefore facilitate the analysis by MALDI-MS. This C-terminal sequence analysis method is also useful for large proteins after fragmentation with specific enzymes.

Amino Acid Sequence↗

PubMed1994

2.8-A structure of yeast serine carboxypeptidase.

The structure of monomeric serine carboxypeptidase from Saccharomyces cerevisiae (CPD-Y), deglycosylated by an efficient new procedure, has been determined by multiple isomorphous replacement and crystallographic refinement. The model contains 3333 non-hydrogen atoms, all 421 amino acids, 3 of 4 carbohydrate residues, 5 disulfide bridges, and 38 water molecules. The standard crystallographic R-factor is 0.162 for 10,909 reflections observed between 20.0- and 2.8-A resolution. The model has rms deviations from ideality of 0.016 A for bond lengths and 2.7 degrees for bond angles and from restrained thermal parameters of 7.9 A2. CPD-Y, which exhibits a preference for hydrophobic peptides, is distantly related to dimeric wheat serine carboxypeptidase II (CPD-WII), which has a preference for basic peptides. Comparison of the two structures suggests that substitution of hydrophobic residues in CPD-Y for negatively charged residues in CPD-WII in the binding site is largely responsible for this difference. Catalytic residues are in essentially identical configurations in the two molecules, including strained main-chain conformational angles for three active site residues (Ser 146, Gly 52, and Gly 53) and an unusual hydrogen bond between the carboxyl groups of Glu 145 and Glu 65. The binding of an inhibitor, benzylsuccinic acid, suggests that the C-terminal carboxylate binding site for peptide substrates is Asn 51, Gly 52, Glu 145, and His 397 and that the "oxyanion hole" consists of the amides of Gly 53 and Tyr 147. A surprising result of the study is that the domains consisting of residues 180-317, which form a largely alpha-helical insertion into the highly conserved cores surrounding the active site, are quite different structurally in the two molecules. It is suggested that these domains have evolved much more rapidly than other parts of the molecule and are involved in substrate recognition.

Amino Acid Sequence↗

PubMed1994

Structure of the complex of L-benzylsuccinate with wheat serine carboxypeptidase II at 2.0-A resolution.

The structure of the complex of L-benzylsuccinate (Ki = 0.2 mM) bound to wheat serine carboxypeptidase II has been analyzed at 2.0-A resolution for native and inhibited crystals at -170 degrees C. The model has been refined and has a standard crystallographic R-factor of 0.176 for 57,734 reflections observed between 20.0- and 2.0-A resolution. The root mean square deviation from ideal bonds is 0.017 A and from ideal angles is 2.6 degrees. The model consists of 400 amino acids, 4 N-linked saccharide residues, and 430 water molecules. L-Benzylsuccinate occupies a narrow slot in the active site defined by Tyr 60, Tyr 239, and the polypeptide backbone. One carboxylate forms hydrogen bonds to Glu 145, Asn 51, the amide of Gly 52, and the catalytic His 397, suggestive of how the peptide C-terminal carboxylate is recognized by the enzyme. The phenyl ring stacks between Tyr 239 and Tyr 60, while the other carboxylate occupies the "oxyanion hole". One of the oxygens accepts hydrogen bonds from the amides of Tyr 147 and Gly 53, while the other forms a very close contact (2.3 A) with the O gamma of Ser 146, forcing the side chain into a conformation alternative to that found in the resting state of the enzyme. The inhibitor occupies the active site in a way that suggests that it can be regarded as a transition-state analogue of serine carboxypeptidases. The model suggests a novel enzymatic mechanism, involving substrate-assisted catalysis, that might account for the low pH optimum (4.0-5.5) of peptidase activity unique to this family of serine proteinases.

Amino Acid Sequence↗

PubMed1990

Fragmentation of isoaspartyl peptides and proteins by carboxypeptidase Y: release of isoaspartyl dipeptides as a result of internal and external cleavage.

Isoaspartate-containing versions of sea urchin sperm-activating peptide, delta sleep-inducing peptide, and lactate dehydrogenase (231-242) were cleaved at internal sites by carboxypeptidase Y. Cleavage occurred between the isoaspartate and the preceding amino acid, and it was accompanied by sequential digestion of amino acids from the two resulting carboxyl termini. Because the isoaspartyl bonds were not cleaved, isoaspartyl dipeptides were among the final products. The rate of release of isoaspartyl dipeptides was different for the three peptides, a 24-h digestion yielding 0.32 mol of isoaspartylglycine/mol of isoaspartyl sperm-activating peptide, 0.50 mol of isoaspartylalanine/mol of isoaspartyl delta sleep-inducing peptide, and 1.15 mol of isoaspartylserine/mol of isoaspartyl lactate dehydrogenase (231-242). The different rates could be explained by the slow cleavage of amino acids preceded by glycine. Isoaspartyl dipeptides were not detected in digests of the corresponding aspartate- or asparagine-containing forms of the peptides. Release of isoaspartyl dipeptides by carboxypeptidase Y was used to demonstrate the presence of isoaspartylglycine sequences in deamidated adrenocorticotropin (0.54 mol/mol), in a mixture of trypic fragments of base-treated calmodulin (0.20 mol/mol), and in a mixture of tryptic fragments of base-treated triosephosphate isomerase (0.08 mol/mol). These results confirm earlier work suggesting that isoaspartylglycine formation is prevalent in proteins exposed to alkaline conditions. They also provide a methodology that should prove useful in the characterization of natural substrates for protein L-isoaspartyl methyltransferase.

Animals↗

PubMed1976

Chemical modification of carboxypeptidase A crystals. Nitration of tyrosine-248.

Nitration of bovine carboxypeptidase A crystals with tetranitromethane increases esterase activity, decreases peptidase activity, and modifies about one tyrosyl residue. Modification of enzyme crystals avoids the polymerization that occurs when the enzyme is nitrated in solution. Two procedures have been employed to identify the tyrosyl residues nitrated. The first involves cyanogen bromide cleavage and isolation of the fragment containing residues 104-301. After solubilization by succinylation, this fragment is digested with chymotrypsin, the peptides are fractionated by gel filtration, and the nitrotyrosyl peptides are purified by affinity chromatography on an antinitrotyrosyl antibody-Sepharose conjugate followed by ion-exchange chromatography. In the second, the nitroenzyme is heat denatured, digested by chymotrypsin, and fractionated on the affinity and ion-exchange columns. By both methods, the major mitropeptides, representing between 60 and 80% of the nitrotyrosyl label, are uniquely compatible with that segment of the sequence of carboxypeptidase containing Tyr-248. A nearby cation, either the active site zinc ion or Arg-145, would seem to be an important factor in determining the selective nitration of this residue.

Amino Acids↗

PubMed1975

Immobilized carboxypeptidase A as a probe for studying the thermally induced unfolding of bovine pancreatic ribonuclease.

A method for the preparation of Sephadex-immobilized carboxypeptidase A is presented. This form of the enzyme has the same specific activity as the soluble enzyme at room temperature, but retains its activity at higher temperatures (60-70 degrees). This preparation of immobilized carboxypeptidase A was used, as a proteolytic probe, to investigate the thermally induced unfolding of the C-terminus of ribonuclease A. This technique indicates that the C-terminal residues of ribonuclease A do not unfold until the high-temperature region of the thermal transition (as determined by ultraviolet difference spectrophotometry and optical rotation).

Animals↗

PubMed1975

Environment and conformation dependent sensitivity of the arsanilazotyrosine-248 carboxypeptidase A chromophore.

Reaction of carboxypeptidase A crystals with diazotized arsanilic acid uniquely modifies Tyr-248 to form a monazo derivative, which-in solution-forms an intramolecular inner-sphere coordination complex in the active site zinc atom. tarsanilazocarboxypeptidase exhibits spectral properties that are closely similar to those of the model complex, tetrazolylazo-N-carbobenzoxytyrosine Zn2+, with a distinctive maximum at 510 nm. In addition, its circular dichroic spectrum reveals a negative extremum at this wavelength, also characteristic of this complex. Both spectra are exquisitely responsive to pth changes and serve to monitor formation and dissociation of the metal-azophenol complex. Two pKapp at 7.7 and 9.5 delineate the pH range over which the probe characteristics most effectively gauge conformational features of the active center of arsanilazcarboxypeptidase. Other environmental parameters, e.g., substrates and inhibitors, as well as crystallization of the enzyme also critically influence the formation and dissociation of the complex; the response of the probe suggests that they induce conformational movement of the azoTyr-248 residue away from the zinc atom. tthe now available chemical, functional, structural data bearing on the spatial relationships of Tyr-248 and Zn, both thought critical to catalysis, are evaluated, based on spectra of arsanilazo- and nitrocarboxypeptidase crystals and solutions as well as on detailed kinetic analyses of the native enzyme in both physical states and based on the X-ray structure analysis of the native enzyme and its Gly-L-Tyr complex. Collectively all of the data show that the conformation of carboxypeptidase in crystals differs from that in solution. Moreover, reexamination of the original X-ray maps reported in 1968 and thought to preclude a Tyr-248-Zn interaction now leads to the conclusion that in up to 25 per cent of the molecules in the crystals ttyr-248 interacts with the active site zinc atom (W.D. Lipscomb (1973), Proc. Nat. Acad. Sci U.S. 70, 3797). Thus, even in the crystals the enzyme exists in at least two different conformations. In one of these Tyr-248 is near while in the other it is far from the zinc atom. The spectral effects of Gly-L-Tyr and beta-phenylpropionate on solutions of arsanilazo- and of nitrocarboxypeptidase demonstrate that during the catalytic process Tyr-248 moves away from the zinc atom. This implies a mechanistic role for Tyr-248 different from that postulated on the basis of X-ray crystallographic analysis. Indeed, the proximity of ttyr-248 to the zinc atom, when altered by substrates and inhibitor, may reflect certain of the properties characteristic of the entatic, active site.

Animals↗

PubMed1975

Gas chromatography-mass spectrometry for probing the structure and mechanism of action of enzyme active sites. The role of Glu-270 in carboxypeptidase A.

A new technique for the study of the mechanism of enzymes has been developed. An enzyme, modified by an active-site directed reagent, is digested by one or more proteases. The resulting mixture of oligopeptides is then analyzed directly by gas chromatography-mass spectrometry without the use of separation or isolation procedures. A comparison with unmodified enzyme identifies the modified residue as well as quantifies the reaction. This approach has been applied to the identification of Glu-270 in the active site of carboxypeptidase A using a carbodiimide as modification reagent. Studies on the possible incorporation of 18O (from 18O-enriched water) into Glu-270 or other acidic residues near the active site of carboxypeptidase A show that the oxygens of the carboxyl groups of these residues are not exchangeable.

Amino Acid Sequence↗

PubMed2003

Neisseria gonorrhoeae penicillin-binding protein 3 exhibits exceptionally high carboxypeptidase and beta-lactam binding activities.

A soluble form of penicillin-binding protein 3 (PBP 3) from Neisseria gonorrhoeae was expressed and purified from Escherichia coli and characterized for its interaction with beta-lactam antibiotics, its catalytic properties with peptide and peptidoglycan substrates, and its role in cell viability and morphology. PBP 3 had an unusually high k(2)/K' value relative to other PBPs for acylation with penicillin (7.7 x 10(5) M(-1) s(-1)) at pH 8.5 at 25 degrees C and hydrolyzed bound antibiotic very slowly (k(3) < 4.6 x 10(-5) s(-1), t(1/2) > 230 min). PBP 3 also demonstrated exceptionally high carboxypeptidase activity with a k(cat) of 580 s(-1) and a k(cat)/K(m) of 1.8 x 10(5) M(-1) s(-1) with the substrate N(alpha)-Boc-N(epsilon)-Cbz-L-Lys-D-Ala-D-Ala. This is the highest k(cat) value yet reported for a PBP or other serine peptidases. Activity against a approximately D-Ala-D-Lac peptide substrate was approximately 2-fold lower than against the analogous approximately D-Ala-D-Ala peptide substrate, indicating that deacylation is rate determining for both amide and ester hydrolysis. The pH dependence profiles of both carboxypeptidase activity and beta-lactam acylation were bell-shaped with maximal activity at pH 8.0-8.5. PBP 3 displayed weak transpeptidase activity in a model transpeptidase reaction but was active as an endopeptidase, cleaving dimeric peptide cross-links. Deletion of PBP 3 alone had little effect on viability, growth rate, and morphology of N. gonorrhoeae, although deletion of both PBP 3 and PBP 4, the other low-molecular-mass PBP in N. gonorrhoeae, resulted in a decreased growth rate and marked morphological abnormalities.

Acylation↗

PubMed2005

Crystal structures of potent thiol-based inhibitors bound to carboxypeptidase B.

This paper presents the crystal structure of porcine pancreatic carboxypeptidase B (pp-CpB) in complex with a variety of thiol-based inhibitors that were developed as antagonists of activated thrombin-activatable fibrinolysis inhibitor (TAFIa). Recent studies have indicated that a selective inhibitor of TAFIa could enhance the efficacy of existing thrombolytic agents for the treatment of acute myocardial infarction, one of the most prevalent forms of heart attacks. Unfortunately, activated TAFIa rapidly degrades in solution and cannot be used for crystallographic studies. In contrast, porcine pancreatic CpB is stable at room temperature and is available from commercial sources. Both pancreatic CpB and TAFIa are zinc-based exopeptidases, and the proteins share a 47% sequence identity. The homology improves considerably in the active site where nearly all of the residues are conserved. The inhibitors used in this study were designed to mimic a C-terminal arginine residue, one of the natural substrates of TAFIa. The X-ray structures show that the thiol group chelates the active site zinc, the carboxylic acid forms a salt bridge to Arg145, and the guanidine group forms two hydrogen bonds to Asp255. A meta-substituted phenyl was introduced into our inhibitors to reduce conformational freedom. This modification vastly improved the selectivity of compounds against other exopeptidases that cleave basic residues. Comparisons between structures indicate that selectivity derives from the interaction between the guanidine group in the inhibitors and an acidic active site residue. The location of this acidic residue is not conserved in the various carboxypeptidases.

Animals↗

PubMed1997

Substrates with charged P1 residues are efficiently hydrolyzed by serine carboxypeptidases when S3-P1 interactions are facilitated.

The high activity of carboxypeptidase S1 with substrates having basic P1 residues is predicted to depend on the size of residue 312 in combination with the presence of a counter-charge in an alpha-helix above the S1 binding pocket. This hypothesis is tested by the construction of 32 mutant forms of carboxypeptidase Y that combines a reduction in size of residue 312 and the introduction of either a basic or an acidic residue at either position 241 or position 245. Kinetic characterization using substrates with Leu, Arg, Lys, Glu, or Asp in P1 demonstrates that most of these enzymes exhibit drastically altered catalytic properties. One mutant enzyme, N241D + W312L, hydrolyzes FA-Arg-Ala-OH with a kcat/KM value of 13 000 min-1 mM-1 corresponding to a 930-fold increase relative to the wild-type enzyme. This increased activity is due to an increase in kcat and is independent of ionic strength. The pH profile of kcat/KM exhibits an optimum around pH 5.5 similar to that observed for CPD-S1. Another mutant enzyme, L245R + W312S, hydrolyzes FA-Glu-Ala-OH and FA-Asp-Ala-OH with kcat/KM values of 5100 and 5300 min-1 mM-1, respectively, corresponding to 120 and 170-fold increases relative to wild-type values. With the latter substrate, a 280-fold reduction of KM is observed. The activity of L245R + W312S is also independent of ionic strength and displays a virtually unaltered dependence on pH. The P1 substrate preference of these two mutant enzymes for Arg versus Asp differs 2.5 x 10(6)-fold. values of single and double mutants demonstrate that the effects of reducing the size of Trp312 and introducing a charged residue at position 241 or 245 in some cases exceed 100% additivity. Thus, the double mutant enzyme gains more activation energy than can be accounted for by each individual single mutation.

Binding Sites↗

PubMed1999

Human cathepsin X: A cysteine protease with unique carboxypeptidase activity.

Cathepsin X is a novel cysteine protease which was identified recently from the EST (expressed sequence tags) database. In a homology model of the mature cathepsin X, a unique three residue insertion between the Gln22 of the oxyanion hole and the active site Cys31 was found to be located in the primed region of the binding cleft as part of a surface loop corresponding to residues His23 to Tyr27, which we have termed the "mini-loop". From the model, it became apparent that this distinctive structural feature might confer exopeptidase activity to the enzyme. To verify this hypothesis, human procathepsin X was expressed in Pichia pastoris and converted to mature cathepsin X using small amounts of human cathepsin L. Cathepsin X was found to display excellent carboxypeptidase activity against the substrate Abz-FRF(4NO(2)), with a k(cat)/K(M) value of 1.23 x 10(5) M(-)(1) s(-)(1) at the optimal pH of 5.0. However, the activity of cathepsin X against the substrates Cbz-FR-MCA and Abz-AFRSAAQ-EDDnp was found to be extremely low, with k(cat)/K(M) values lower than 70 M(-)(1) s(-)(1). Therefore, cathepsin X displays a stricter exopeptidase activity than cathepsin B. No inhibition of cathepsin X by cystatin C could be detected up to a concentration of 4 microM of inhibitor. From a model of the protease complexed with Cbz-FRF, the bound carboxypeptidase substrate is predicted to establish a number of favorable contacts within the cathepsin X binding site, in particular with residues His23 and Tyr27 from the mini-loop. The presence of the mini-loop restricts the accessibility of cystatin C as well as of the endopeptidase and MCA substrates in the primed subsites of the protease. The marked structural and functional differences of cathepsin X relative to other members of the papain family of cysteine proteases will be of great value in designing specific inhibitors useful as research tools to investigate the physiological and potential pathological roles of this novel enzyme.

Carboxypeptidases↗

PubMed2002

Substrate-based design of the first class of angiotensin-converting enzyme-related carboxypeptidase (ACE2) inhibitors.

Angiotensin-converting enzyme-related carboxypeptidase (ACE2) is a recently identified zinc metalloprotease with carboxypeptidase activity that was identified using our genomics platform. We implemented a rational design approach to identify potent and selective ACE2 inhibitors. To this end, picomolar inhibitors of ACE2 were designed and synthesized.

Angiotensin-Converting Enzyme 2↗

PubMed1972

Radiochemical determination of a unique sequence around the reactive serine residue of a di-isopropyl phosphorofluoridate-sensitive plant carboxypeptidase and a yeast peptidase.

Phaseolain, a carboxypeptidase from French-bean leaves, and a partially purified peptidase from baker's yeast are inhibited by reaction with di-isopropyl phosphorofluoridate. Radioactive di-isopropyl [(32)P]phosphorofluoridate was used to show that the site of reaction is a unique serine residue and that the sequence of amino acids adjacent to the reactive serine is Glu-Ser-Tyr. This sequence is different from those of other ;serine' enzymes previously reported and, for phaseolain, represents an unequivocal example of a ;serine' carboxypeptidase.

Amino Acid Sequence↗

PubMed2003

Subsite specificity (S3, S2, S1', S2' and S3') of oligopeptidase B from Trypanosoma cruzi and Trypanosoma brucei using fluorescent quenched peptides: comparative study and identification of specific carboxypeptidase activity.

We characterized the extended substrate binding site of recombinant oligopeptidase B enzymes from Trypanosoma cruzi (Tc-OP) and Trypanosoma brucei (Tb-OP), evaluating the specificity of their S3, S2, S1', S2' and S3' subsites. Five series of internally quenched fluorescent peptides based on the substrate Abz-AGGRGAQ-EDDnp [where Abz is o -aminobenzoic acid and EDDnp is N -(2,4-dinitrophenyl)ethylenediamine] were designed to contain amino acid residues with side chains of a minimum size, and each residue position of this substrate was modified. Synthetic peptides of different lengths derived from the human kininogen sequence were also examined, and peptides of up to 17 amino acids were found to be hydrolysed by Tc-OP and Tb-OP. These two oligopeptidases were essentially arginyl hydrolases, since for all peptides examined the only cleavage site was the Arg-Xaa bond. We also demonstrated that Tc-OP and Tb-OP have a very specific carboxypeptidase activity for basic amino acids, which depends on the presence of at least of a pair of basic amino acids at the C-terminal end of the substrate. The peptide with triple Arg residues (Abz-AGRRRAQ-EDDnp) was an efficient substrate for Tc-OP and Tb-OP: the Arg-Ala peptide bond was cleaved first and then two C-terminal Arg residues were successively removed. The S1' subsite seems to be an important determinant of the specificity of both enzymes, showing a preference for Tyr, Ser, Thr and Gln as hydrogen donors. The presence of these amino acids at P1' resulted in substrates that were hydrolysed with K (m) values in the sub-micromolar range. Taken together, this work supports the view that oligopeptidase B is a specialized protein-processing enzyme with a specific carboxypeptidase activity. Excellent substrates were obtained for Tb-OP and Tc-OP (Abz-AMRRTISQ-EDDnp and Abz-AHKRYSHQ-EDDnp respectively), which were hydrolysed with remarkably high k (cat) and low K (m) values.

Amino Acid Sequence↗