Serine Carboxypeptidase Research Articles

Cellular carboxypeptidases.

This article focuses on four human carboxypeptidases (CPs): two metallo-CPs and two serine CPs. The metallo-CPs are members of the so-called B-type regulatory CP family, as they cleave only the C-terminal basic amino acids Arg or Lys. The plasma membrane-bound CPM and the mainly, but not exclusively, intracellular CPD are surveyed from this group of enzymes. These enzymes can regulate peptide hormone activity at the cell surface and possibly intracellularly after receptor-mediated endocytosis and may also participate in peptide hormone processing. The serine CPs, as their name indicates, contain a serine residue in the active center essential for catalytic activity that reacts with organophosphorus inhibitors. Prolylcarboxypeptidase (PRCP) (angiotensinase C) and deamidase (cathepsin A, lysosomal protective protein) are discussed here. These two enzymes are highly concentrated in lysosomes; however, they may also be active extracellularly after their release from lysosomes in soluble form or in a plasma membrane-bound complex. Whereas deamidase cleaves a variety of peptides with C-terminal or penultimate hydrophobic residues (e.g. substance P, angiotensin I, bradykinin, endothelin, fMet-Leu-Phe). PRCP cleaves only peptides with a penultimate Pro residue (e.g. des-Arg9-bradykinin, angiotensin II). These enzymes may also be involved in terminating signal transduction by inactivating peptide ligands after receptor endocytosis.

Separation of proteolytic enzymes originating from Antarctic krill ( Euphausia superba) by capillary electrophoresis

Extracts prepared from Antarctic krill ( Euphausia superba), mainly consisting of acidic proteolytic enzymes, have been studied with capillary electrophoretic techniques. Approximately 50 repeatable peaks were obtained with capillary zone electrophoresis on an untreated fused-silica capillary using a phosphate buffer containing anionic and cationic fluorosurfactant additives as separation medium. A faster separation was achieved on a polyvinyl alcohol coated capillary. Quantitative variations of individual proteins regarding different krill enzyme batches were noted. In the krill samples trypsin-like serine proteinase, carboxypeptidase A and carboxypeptidase B were tentatively identified.

The atomic model of the human protective protein/cathepsin A suggests a structural basis for galactosialidosis.

Human protective protein/cathepsin A (PPCA), a serine carboxypeptidase, forms a multienzyme complex with beta-galactosidase and neuraminidase and is required for the intralysosomal activity and stability of these two glycosidases. Genetic lesions in PPCA lead to a deficiency of beta-galactosidase and neuraminidase that is manifest as the autosomal recessive lysosomal storage disorder galactosialidosis. Eleven amino acid substitutions identified in mutant PPCAs from clinically different galactosialidosis patients have now been modeled in the three-dimensional structure of the wild-type enzyme. Of these substitutions, 9 are located in positions likely to alter drastically the folding and stability of the variant protein. In contrast, the other 2 mutations that are associated with a more moderate clinical outcome and are characterized by residual mature protein appeared to have a milder effect on protein structure. Remarkably, none of the mutations occurred in the active site or at the protein surface, which would have disrupted the catalytic activity or protective function. Instead, analysis of the 11 mutations revealed a substantive correlation between the effect of the amino acid substitution on the integrity of protein structure and the general severity of the clinical phenotype. The high incidence of PPCA folding mutants in galactosialidosis reflects the fact that a single point mutation is unlikely to affect both the beta-galactosidase and the neuraminidase binding sites of PPCA at the same time to produce the double glycosidase deficiency. Mutations in PPCA that result in defective folding, however, disrupt every function of PPCA simultaneously.

Structure of proline iminopeptidase from Xanthomonas campestris pv. citri: a prototype for the prolyl oligopeptidase family.

The proline iminopeptidase from Xanthomonas campestris pv. citri is a serine peptidase that catalyses the removal of N-terminal proline residues from peptides with high specificity. We have solved its three-dimensional structure by multiple isomorphous replacement and refined it to a crystallographic R-factor of 19.2% using X-ray data to 2.7 A resolution. The protein is folded into two contiguous domains. The larger domain shows the general topology of the alpha/beta hydrolase fold, with a central eight-stranded beta-sheet flanked by two helices and the 11 N-terminal residues on one side, and by four helices on the other side. The smaller domain is placed on top of the larger domain and essentially consists of six helices. The active site, located at the end of a deep pocket at the interface between both domains, includes a catalytic triad of Ser110, Asp266 and His294. Cys269, located at the bottom of the active site very close to the catalytic triad, presumably accounts for the inhibition by thiol-specific reagents. The overall topology of this iminopeptidase is very similar to that of yeast serine carboxypeptidase. The striking secondary structure similarity to human lymphocytic prolyl oligopeptidase and dipeptidyl peptidase IV makes this proline iminopeptidase structure a suitable model for the three-dimensional structure of other peptidases of this family.

Action of serine carboxypeptidase from paecilomyces carneus on oligopeptides containing carboxy-terminally amidated peptides

Paecilomyces carneus carboxypeptidase sequentially liberated amino acids from the carboxy-terminus of neurotensin, angiotensin I, bradykinin, and delta sleep-inducing peptide, indicating that the sequential hydrolysis of peptides was limited by the occurrence of intermediates with the structure of -Gly-X (X = L-amino acid), -Pro-X, -X-Gly, and -X-Pro. The enzyme had carboxyamidase and/or amidase activities for the carboxy-terminally amidated peptides. The enzyme essentially acted as a carboxyamidase for the long carboxy-terminally amidated peptides; an amidase became dominant for the substrates in the presence of bulky amino acids such as Arg, Met, Leu, and Phe in the penultimate (P1) and P2 positions, corresponding with the S1 and S2 sites of the enzyme, and the P3 position of carboxy-terminally amidated peptides played a significant role in the action as a carboxyamidase or a amidase.

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.

Crystal structure of Kex1deltap, a prohormone-processing carboxypeptidase from Saccharomyces cerevisiae,.

Kex1p is a prohormone-processing serine carboxypeptidase found in Saccharomyces cerevisiae. In contrast to yeast serine carboxypeptidase (CPD-Y) and wheat serine carboxypeptidase II (CPDW-II), Kex1p displays a very narrow specificity for lysyl or arginyl residues at the C-terminus of the substrate. The structure of Kex1Deltap, an enzyme that lacks the acidic domain and membrane-spanning portion of Kex1p, has been solved by a combination of molecular replacement and multiple isomorphous replacement and refined to a resolution of 2.4 A. The S1' site of Kex1Deltap is sterically restricted compared to those from CPD-Y or CPDW-II; it also contains two acidic groups that are well positioned to interact with the basic group of a lysine or arginine side chain. The high specificity of Kex1p can therefore be explained by a combination of steric and electronic factors. The structure of the S1 site of Kex1Deltap is also well suited for binding of a lysine or arginine side chain, and the enzyme may therefore exhibit a preference for these residues at P1.

Vacuolar protein sorting in fission yeast: cloning, biosynthesis, transport, and processing of carboxypeptidase Y from Schizosaccharomyces pombe.

PCR was used to isolate a carboxypeptidase Y (CPY) homolog gene from the fission yeast Schizosaccharomyces pombe. The cloned S. pombe cpy1+ gene has a single open reading frame, which encodes 950 amino acids with one potential N-glycosylation site. It appears to be synthesized as an inactive pre-pro protein that likely undergoes processing following translocation into appropriate intracellular organelles. The C-terminal mature region is highly conserved in other serine carboxypeptidases. In contrast, the N-terminal pro region containing the vacuolar sorting signal in CPY from Saccharomyces cerevisiae shows fewer identical residues. The pro region contains two unusual repeating sequences; repeating sequence I consists of seven contiguous repeating segments of 13 amino acids each, and repeating sequence II consists of seven contiguous repeating segments of 9 amino acids each. Pulse-chase radiolabeling analysis revealed that Cpy1p was initially synthesized in a 110-kDa pro-precursor form and via the 51-kDa single-polypeptide-chain intermediate form which has had its pro segment removed is finally converted to a heterodimer, the mature form, which is detected as a 32-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Like S. cerevisiae CPY, S. pombe Cpy1p does not require the N-linked oligosaccharide moiety for vacuolar delivery. To investigate the vacuolar sorting signal of S. pombe Cpy1p, we have constructed cpy1+-SUC2 gene fusions that direct the synthesis of hybrid proteins consisting of N-terminal segments of various lengths of S. pombe Cpy1p fused to the secreted enzyme S. cerevisiae invertase. The N-terminal 478 amino acids of Cpy1 are sufficient to direct delivery of a Cpy1-Inv hybrid protein to the vacuole. These results showed that the pro peptide of Cpy1 contains the putative vacuolar sorting signal.

Disruption of three acid proteases in Aspergillus niger--effects on protease spectrum, intracellular proteolysis, and degradation of target proteins.

Three acid protease genes encoding two extracellular proteases (PEPA and PEPB) and one intracellular protease (PEPE) were disrupted in Aspergillus niger. Northern-blot analysis showed the absence of wild-type protease mRNAs in the disruptants while western-blot analysis proved the absence of the encoded proteases. Characterization of the residual proteolytic spectra in the disruptants indicated that the extracellular protease activity was reduced to 16% and 94% for the delta pepA and the delta pepB disruptants, repectively. In the delta pepE disruptant, the total intracellular proteolytic activity was reduced to 32%. Apart from the reduced intracellular pepstatin-inhibitable aspartyl protease activity, serine protease and serine carboxypeptidase activities were also significantly reduced in the delta pepE strain. This may indicate the presence of a cascade activation mechanism for several vacuolar proteases, triggered by the PEPE protein, similar to the situation in Saccharomyces cerevisiae. Disruption of a single protease gene had no effects on the transcription of other non-disrupted protease genes in A. niger. In supernatants of the disruptants, reduced degradation of a proteolytically very susceptible tester protein (PELB) was observed. By recombination, we also constructed delta pepA delta pepB, delta pepB delta pepE and delta pepA delta pepE double disruptants as well as a delta pepA delta pepB delta pepE triple disruptant, lacking all three acid protease activities. The in vitro residual PELB activity was the highest in the triple disruptant and the delta pepA delta pepB recombinant.

Identification of the promoters for the human and murine protective protein/cathepsin A genes.

Protective protein/cathepsin A (PPCA) is a lysosomal serine carboxypeptidase that forms a complex with beta-galactosidase and neuraminidase. Its deficiency in humans leads to the lysosomal storage disorder galactosialidosis (GS). The pathologic manifestations in patients relate primarily to the severe deficiency of neuraminidase, and the physiological significance of cathepsin A activity remains unclear. The mouse model of GS, which closely resembles the human phenotype, shows that cells from numerous tissues, especially the central nervous system (CNS), are affected by this disease. To study the site and level of expression of PPCA mRNA in murine and human tissues, we analyzed the promoter regions of the corresponding genes. Their 5' genomic regions were strikingly similar in both organization and sequence. A single 1.8-kb PPCA transcript is present in humans, whereas mouse tissues have a major 1.8-kb and a minor 2.0-kb transcript, both of which are differentially expressed. These two mouse mRNA species differ only in their 5' untranslated region (UTR). The larger mRNA, unique to mouse, is transcribed from an upstream TATA-box-containing promoter, which is absent in the human gene. The downstream promoter, which transcribes the 1.8-kb mRNA common to human and mouse, has characteristics of housekeeping gene promoters and contains putative Sp1 binding sites and three USF/MLTF sequences. In vitro studies demonstrated that expression from the downstream promoter is higher than that from the upstream murine-specific promoter. In situ hybridization of mouse tissue sections identified regions of the brain that preferentially express the 2.0-kb transcript. Our results imply that PPCA mRNA distribution and regulation in murine tissues differs from that in human tissues.

Structure determination of the human protective protein: twofold averaging reveals the three-dimensional structure of a domain which was entirely absent in the initial model.

Mutations in the human 'protective protein' result in the human lysosomal storage disease galactosialidosis. The structure of the human 'protective protein' has been determined using X-ray crystallography to a resolution of 2.2 A. Initial phases were obtained from molecular replacement calculations. A very partial search model comprising 30% of the scattering mass, was constructed from the atomic model of the wheat serine carboxypeptidase. This truncated probe was used to find the position of two monomers in the asymmetric unit. Subsequently, 'bootstrapping' cycles, consisting of twofold averaging and model expansion, retrieved the electron density for residues initially missing. In particular, it proved possible to add a domain (more than 110 residues) to the initial partial search model. In total, 314 residues per asymmetric unit were added to the 588 residues of the initial model. Factors contributing to our success are discussed.

Production, purification, and properties of serine carboxypeptidase from Paecilomyces carneus.

Seventeen strains of the genus Paecilomyces were examined for their ability to produce serine carboxypeptidase. Paecilomyces carneus IFO 7012 exhibited the highest potency for serine carboxypeptidase production. A maximum yield of serine carboxypeptidase was obtained by koji culture of the strain at 22 degrees C for 7 days. The serine carboxypeptidase was purified to homogeneity from an extract of the koji culture. The molecular weight of the enzyme was estimated to be 47,000 by HPLC. The isoelectric point of the enzyme was determined to be 4.0, and the optimum pH was 4.0 toward benzyloxycarbonyl-L-glutamyl-L-tyrosine (Z-Glu-Tyr) and benzyloxycarbonyl-L-phenylalanyl-L-alanine (Z-Phe-Ala), respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and p-chloromercurybenzoate. Relative hydrolysis rates of N-acylpeptides and kinetic studies indicated that the enzyme preferred substrates having bulky amino acids in the penultimate position from their carboxy-termini.

Evaluation of the Significance of Charge and Length of Side Chain on the P’1- S'1 Interactions In (Serine) Carboxypeptidase Y

Abstract: Many enzymes exhibit a very broad specificity due to the ability of some subsites to accommodate both polar and apolar amino acid residues. In the present study the origin of the ability of CPD-Y to cleave substrates with large apolar as well as positively charged P; amino acid residues has been investigated by utilization of substrates containing amino acids with side chains of varying length and charge.

Protease inhibitor studies and cloning of a serine carboxypeptidase cDNA from germinating seeds of pea (Pisum sativum L.).

The nature of the proteolytic activity found within the germinating pea (Pisum sativum) seed, 4 days from the initiation of imbibition, was determined by the use of specific protease inhibitors. These studies have shown most of the activity to belong to metallo or metal-activated and serine proteases. In order to investigate further the serine protease activity, a pea cotyledon germination cDNA library was, therefore, screened with a wheat cDNA (2437) [Baulcombe, D.C., Barker, R.F. & Jarvis, M.G. (1987) J. Biol. Chem. 262, 13726-13735] which had extensive similarity to the yeast serine carboxypeptidase Y gene. A positive cDNA clone (pNY551) was obtained which had extensive similarity to the four carboxypeptidases, Arabidopsis thaliana carboxypeptidase Y-like protein, rice serine carboxypeptidase III, barley serine carboxypeptidase III and wheat serine carboxypeptidase III precursor. Northern-blot analysis showed mRNA homologous to pNY551 to be expressed in late developmental pea seed and again during germination.

Crystallization of a soluble form of the Kex1p serine carboxypeptidase from Saccharomyces cerevisiae.

A soluble form of the killer factor and prohormone-processing carboxypeptidase, "Kex1 delta p," from Saccharomyces cerevisiae, has been crystallized in 17-22% poly(enthylene glycol) methyl ether (average M(r) = 5,000), 100 mM ammonium acetate, 5% glycerol, pH 6.5, at 20 degrees C. A native data set (2.8 A resolution) and four derivative data sets (3.0-3.2 A resolution) were collected at the Photon Factory (lambda = 1.0 A). The crystals belong to space group P2(1)2(1)2(1) with a =56.6 A, b = 84.0 A, c = 111.8 A. Freezing a Kex1 delta p crystal has facilitated the collection of a 2.4-A data set using a rotating anode source (lambda = 1.5418 A). Molecular replacement models have been built based on the structures of wheat serine carboxypeptidase (CPDW-II; Liao DI et al., 1992, Biochemistry 31:9796-9812) and yeast carboxypeptidase Y.

Peptide Aldehyde Complexes with Wheat Serine Carboxypeptidase II: Implications for the Catalytic Mechanism and Substrate Specificity

The structures of two ternary complexes of wheat serine carboxypeptidase II (CPD-WII), with a tetrapeptide aldehyde and a reaction product arginine, have been determined by X-ray crystallography at room temperature and −170°. The peptide aldehydes, antipain and chymostatin, form covalent adducts with the active-site serine 146. The CPD-WII antipain arginine model has a standard crystallographic R-factor of 0.162, with good geometry at 2.5 Å resolution for data collected at room temperature. The −170°C model of the chymostatin arginine complex has an R-factor of 0.174, with good geometry using data to 2.1 Å resolution. The structures suggest binding subsites N-terminal to the scissile bond. All four residues of chymostatin are well-localized in the putative S 1through S 4sites, while density is apparent only in S 1and S 2for antipain. In the S 1site, Val340 and 341, Phe215 and Leu216 form a hydrophobic binding surface, not a pocket, for the P 1phenylalanyl side-chain of chymostatin. The P 1arginyl of antipain also binds at this site, but the positive charge appears to be stabilized by additional solvent molecules. Thus, the hybrid nature of the S 1site accounts for the ability of CPD-WII to accept both hydrophobic and basic residues at P 1. Hydrogen bonds to the peptide substrate backbone are few and are made primarily with side-chains on the enzyme. Thus, substrate recognition by CPD-WII appears to have nothing in common with that of the other families of serine proteinases. The hemiacetal linkages to the essential Ser146 are of a single stereoisomer with tetrahedral geometry, with an oxygen atom occupying the "oxyanion hole" region of the enzyme. This atom accepts three hydrogen bonds, two from the polypeptide backbone and one from the positively-charged amino group of bound arginine, and must be negatively charged. Thus, the combination of ligands forms an excellent approxi mation to the oxyanion intermediate formed during peptide hydrolysis. Surprisingly, the (R) stereochemistry at the hemiacetal linkage is opposite to that expected by comparison to previously determined structures of peptide aldehydes complexed with Streptomyces griseusproteinase A. This is shown to be a consequence of the approximate mirror symmetry of the arrangement of catalytic groups in the two families of serine proteases and suggests that the stereochemical course of the two enzymatic reactions differ in handedness.

Comparative modeling of substrate binding in the S1' subsite of serine carboxypeptidases from yeast, wheat, and human.

Human cathepsin A ("lysosomal protective protein"; E.C.3.4.16.5) is a multifunctional lysosomal protein which forms a high-molecular-weight complex with beta-galactosidase and alpha-neuraminidase, protecting them against intralysosomal proteolysis. In addition to this protective function, cathepsin A is a serine carboxypeptidase and the understanding of its catalytic function requires a definition of its substrate specificity. For this purpose, we used a combined experimental [Pshezhetsky, A. V., Vinogradova, M. V., Elsliger, M.-A., El-Zein, F., Svedas, V.K., & Potier, M. (1995) Anal. Biochem. 230, 303-307] and theoretical approach comparing cathepsin A to two different homologous carboxypeptidases of the same family: yeast carboxypeptidase Y and wheat carboxypeptidase II. We computed the energies involved in substrate binding to the S1' subsite (C-terminal) of cathepsin A using a structural model based on the X-ray structure of the homologous wheat carboxypeptidase II. The binding energies of N-blocked Phe-Xaa dipeptide substrates to the active sites of cathepsin A, wheat carboxypeptidase II, and yeast carboxypeptidase Y were estimated using a molecular mechanics force field supplemented with a solvation energy term. This theoretical analysis showed a good correlation with the experimentally determined free energies of substrate binding. This result validates the use of this approach to analyze the energetics of substrate binding to the S1' subsite and provides a rational interpretation of serine carboxypeptidase-substrate interactions in molecular terms. We conclude that the three serine carboxypeptidases have similar affinities for substrates with hydrophobic P1' amino acid residues but that the wheat enzyme has an additional capacity for binding positively charged P1' residues. Finally, the substrate specificity of human cathepsin A is very similar to that of carboxypeptidase Y, with a high binding affinity for substrates with hydrophobic P1' residues, but the affinity of cathepsin A for P1; Phe residue is higher than for the Leu residue.

Piperastatin A, a new selective serine carboxypeptidase inhibitor produced by actinomycete. I. Taxonomy, production, isolation and biological activities.

Piperastatin A (structure, N-formyl-allo Ile-Thr-Leu-Val-Pip-Leu-Pip, Pip = hexahydropyridadine-3-carboxylic acid; molecular weight, 809), a new inhibitor of serine carboxypeptidase was discovered in the fermentation broth of Streptomyces lavendofoliae MJ908-WF13. It was purified by activated charcoal chromatography, YMC gel ODS-A chromatography and centrifugal partition chromatography (CPC) by monitoring its inhibitory activity against carboxypeptidase Y (CP-Y), and finally obtained as colourless needles. Piperastatin A is a competitive inhibitor of the enzyme with Ki = 52 +/- 6.2 nM. Piperastatin A is a highly specific inhibitor of the serine carboxypeptidases, CP-Y and platelet deamidase with little effect on related enzymes, has no antimicrobial activity and has low toxicity.

Studies on the hydrolytic properties of (serine) carboxypeptidase Y.

The activity of serine carboxypeptidases is dependent on a catalytic triad, an oxyanion hole, and a binding site equivalent to those found in the serine endopeptidases. The action of carboxypeptidase Y on substrates containing amino acids, alcohols, and amines as leaving groups is described. It is demonstrated that the features common to serine endopeptidases and carboxypeptidases are sufficient for hydrolysis of ester bonds. However, rapid hydrolysis of amide bonds is dependent on interactions between the C-terminal carboxylate group of the substrate and the C-terminal recognition site of the enzyme. Furthermore, on the basis of the pH dependencies of wild-type and mutant enzyme, combined with the ability of the enzyme to utilize binding energy to promote catalysis, alternative models for the high activity of carboxypeptidase Y at low pH are discussed. They describe how the catalytically essential histidine is maintained in its active deprotonated state through perturbation of its pKa value in the enzyme-substrate complex.

Piperastatin B: a new selective serine carboxypeptidase inhibitor from Streptomyces lavendofoliae MJ908-WF13.

Piperastatin B, a new inhibitor of serine carboxypeptidase was purified from a culture broth of Streptomyces lavendofoliae MJ908-WF13 as a minor component by monitoring its inhibitory activity against carboxypeptidase Y (CP-Y). Its structure was determined to be N-formyl-Val-Thr-Leu-Val-Pip-Leu-Pip (pip: piperazic acid, hexahydropyridadine-3-carboxylic acid). Piperastatin B is a highly specific competitive inhibitor of CP-Y (Ki = 55 nM) with little effect on related enzymes and resembles the major component, piperastatin A, in these respects.