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Metallocarboxypeptidase Z (CPZ) is a secreted enzyme that is distinguished from all other members of the M14 metallocarboxypeptidase family by the presence of an N-terminal cysteine-rich Frizzled-like (Fz) domain that binds Wnt proteins. Here, we present a comprehensive analysis of the enzymatic properties and substrate specificity of human CPZ. To investigate the enzymatic properties, we employed dansylated peptide substrates. For substrate specificity profiling, we generated two different large peptide libraries and employed isotopic labeling and quantitative mass spectrometry to study the substrate preference of this enzyme. Our findings revealed that CPZ has a strict requirement for substrates with C-terminal Arg or Lys at the P1&prime, position. For the P1 position, CPZ was found to display specificity towards substrates with basic, small hydrophobic, or polar uncharged side chains. Deletion of the Fz domain did not affect CPZ activity as a carboxypeptidase. Finally, we modeled the structure of the Fz and catalytic domains of CPZ. Taken together, these studies provide the molecular elucidation of substrate recognition and specificity of the CPZ catalytic domain, as well as important insights into how the Fz domain binds Wnt proteins to modulate their functions.

In the current study we compared the molecular signature of expanded mesenchymal stromal cells (MSCs) derived from selected CD271+ bone marrow mononuclear cells (CD271-MSCs) and MSCs derived from non-selected bone marrow mononuclear cells by plastic adherence (PA-MSCs). Transcriptome analysis demonstrated for the first time the upregulation of 115 and downregulation of 131 genes in CD271-MSCs. Functional enrichment analysis showed that the upregulated genes in CD271-MSCs are significantly enriched for extracellular matrix (tenascin XB, elastin, ABI family, member 3 (NESH) binding protein, carboxypeptidase Z, laminin alpha 2 and nephroblastoma overexpressed) and cell adhesion (CXCR7, GPNMB, MYBPH, SVEP1, ARHGAP6, TSPEAR, PIK3CG, ABL2 and NCAM1). CD271-MSCs expressed higher gene transcript levels that are involved in early osteogenesis/chondrogenesis/adipogenesis (ZNF145, FKBP5). In addition, increased transcript levels for early and late osteogenesis (DPT, OMD, ID4, CRYAB, SORT1), adipogenesis (CTNNB1, ZEB, LPL, FABP4, PDK4, ACDC), and chondrogenesis (CCN3/NOV, CCN4/WISP1, CCN5/WISP2 and ADAMTS-5) were detected. Interestingly, CD271-MSCs expressed increased levels of hematopoiesis associated genes (CXCL12, FLT3L, IL-3, TPO, KITL). Down-regulated genes in CD271-MSCs were associated with WNT and TGF-beta signaling, and cytokine/chemokine signaling pathways. In addition to their capacity to support hematopoiesis, these results suggest that CD271-MSCs may contain more osteo/chondro progenitors and/or feature a greater differentiation potential.

Frizzleds (FZDs) are transmembrane receptors in the Wnt signaling pathway and they play pivotal roles in developments. The Frizzled-like extracellular Cysteine-rich domain (Fz-CRD) has been identified in FZDs and other proteins. The origin and evolution of these proteins with Fz-CRD is the main interest of this study. We found that the Fz-CRD exists in FZD, SFRP, RTK, MFRP, CPZ, CORIN, COL18A1 and other proteins. Our systematic analysis revealed that the Fz-CRD domain might have originated in protists and then fused with the Frizzled-like seven-transmembrane domain (7TM) to form the FZD receptors, which duplicated and diversified into about 11 members in Vertebrates. The SFRPs and RTKs with the Fz-CRD were found in sponge and expanded in Vertebrates. Other proteins with Fz-CRD may have emerged during Vertebrate evolution through domain fusion. Moreover, we found a glycosylation site and several conserved motifs in FZDs, which may be related to Wnt interaction. Based on these results, we proposed a model showing that the domain fusion and expansion of Fz-CRD genes occurred in Metazoa and Vertebrates. Our study may help to pave the way for further research on the conservation and diversification of Wnt signaling functions during evolution.

Neuroblastoma is a cancer of the developing sympathetic nervous system that most commonly presents in young children and accounts for approximately 12% of pediatric oncology deaths. Here, we report on a genome-wide association study (GWAS) in a discovery cohort or 2,101 cases and 4,202 controls of European ancestry. We identify two new association signals at 3q25 and 4p16 that replicated robustly in multiple independent cohorts comprising 1,163 cases and 4,396 controls (3q25: rs6441201 combined P = 1.2x10-11, Odds Ratio 1.23, 95% CI:1.16–1.31; 4p16: rs3796727 combined P = 1.26x10-12, Odds Ratio 1.30, 95% CI: 1.21–1.40). The 4p16 signal maps within the carboxypeptidase Z (CPZ) gene. The 3q25 signal resides within the arginine/serine-rich coiled-coil 1 (RSRC1) gene and upstream of the myeloid leukemia factor 1 (MLF1) gene. Increased expression of MLF1 was observed in neuroblastoma cells homozygous for the rs6441201 risk allele (P = 0.02), and significant growth inhibition was observed upon depletion of MLF1 (P < 0.0001) in neuroblastoma cells. Taken together, we show that common DNA variants within CPZ at 4p16 and upstream of MLF1 at 3q25 influence neuroblastoma susceptibility and MLF1 likely plays an important role in neuroblastoma tumorigenesis., Author summary Neuroblastoma is an embryonal tumor of the developing sympathetic nervous system that accounts for 12% of childhood cancer deaths. Approximately 1–2% of cases are inherited in an autosomal dominant fashion. These familial cases often harbor germline mutations in ALK or PHOX2B. However, the vast majority of neuroblastomas appear to arise sporadically. We are studying sporadic neuroblastoma through an ongoing genome-wide association study (GWAS). To date, this effort has identified single nucleotide polymorphisms (SNPs) within or upstream of CASC15 and CASC14, BARD1, LMO1, DUSP12, HSD17B12, DDX4/IL31RA, HACE1, LIN28B, and TP53, along with a common copy number variation (CNV) within NBPF23 at chromosome 1q21.1, each being highly associated with neuroblastoma. Here, we report on genome-wide association study (GWAS) comprising 3,264 neuroblastoma patients and 8,598 control subjects. We identify two new association signals at 3q25 and 4p16 (3q25: rs6441201 combined P = 1.2x10-11, Odds Ratio 1.23, 95% CI:1.16–1.31; 4p16: rs3796727 combined P = 1.26x10-12, Odds Ratio 1.30, 95% CI: 1.21–1.40). The 3q25 signal resides upstream of the MLF1 gene and the 4p16 signal maps to the CPZ gene. We further demonstrate that neuroblastoma cells homozygous for the risk allele at 3q25 express higher levels of MLF1 and that silencing of MLF1 in neuroblastoma cells results in significant growth inhibition.

Carboxypeptidase Z (CPZ) removes carboxyl-terminal basic amino acid residues, particularly arginine residues, from proteins. CPZ contains a cysteine-rich domain (CRD) similar to the CRD found in the frizzled family of Wnt receptors. We have previously shown that thyroid hormone regulates terminal differentiation of growth plate chondrocytes through activation of Wnt-4 expression and Wnt/beta-catenin signaling. The Wnt-4 protein contains a C-terminal arginine residue and binds to CPZ through the CRD. The objective of this study was to determine whether CPZ modulates Wnt/beta-catenin signaling and terminal differentiation of growth plate chondrocytes. Our results show that CPZ and Wnt-4 mRNA are co-expressed throughout growth plate cartilage. In primary pellet cultures of rat growth plate chondrocytes, thyroid hormone increases both Wnt-4 and CPZ expression, as well as CPZ enzymatic activity. Knockdown of either Wnt-4 or CPZ mRNA levels using an RNA interference technique or blocking CPZ enzymatic activity with the carboxypeptidase inhibitor GEMSA reduces the thyroid hormone effect on both alkaline phosphatase activity and Col10a1 mRNA expression. Adenoviral overexpression of CPZ activates Wnt/beta-catenin signaling and promotes the terminal differentiation of growth plate cells. Overexpression of CPZ in growth plate chondrocytes also removes the C-terminal arginine residue from a synthetic peptide consisting of the carboxyl-terminal 16 amino acids of the Wnt-4 protein. Removal of the C-terminal arginine residue of Wnt-4 by site-directed mutagenesis enhances the positive effect of Wnt-4 on terminal differentiation. These data indicate that thyroid hormone may regulate terminal differentiation of growth plate chondrocytes in part by modulating Wnt signaling pathways through the induction of CPZ and subsequent CPZ-enhanced activation of Wnt-4.

Carboxypeptidase Z (CPZ) is a secreted Zn-dependent enzyme whose biological function is largely unknown. CPZ has a bipartite structure consisting of an N-terminal cysteine-rich domain (CRD) and a C-terminal catalytic domain. In the early chicken embryo CPZ is initially expressed throughout the somites and subsequently becomes restricted to the sclerotome. To initiate a functional analysis of CPZ, a CPZ producing retroviral vector was applied to the presomitic mesoderm at the level of the future wing. This resulted in a loss of the scapular blade and of rostral ribs. Such dysmorphogenesis is preceded by ectopic Pax3 expression in the hypaxial part of the dermomyotome,a region from which the blade of the scapula normally derives. A mutant CPZ,lacking a critical active site glutamate, fails to induce Pax3expression and does not cause skeletal defects. The induction of Pax3, a Wnt-responsive gene in somites, and the presence of a CRD prompted us to examine whether CPZ affects Wnt signaling. In an in vitro assay we found that CPZ, but not its inactive mutant form, enhances the Wnt-dependent induction of the homeobox gene Cdx1. In addition,immunoprecipitation experiments suggest that the CRD of CPZ acts as a binding domain for Wnt. Taken together these data provide the first evidence for CPZ playing a role in Wnt signaling.

Metallocarboxypeptidase Z (CPZ), a new member of the regulatory metallocarboxypeptidases, contains a 120-residue cysteine-rich region that has 20–35% amino acid sequence identity to Drosophila and mammalian frizzled proteins. In order to gain insights into the function of CPZ, we have examined the distribution of the protein by immunohistochemistry throughout mouse development. The expression of CPZ peaks at E9–E12, decreases in late gestation and falls further in adult tissues. CPZ expression in amnion cells, cochlear epithelial cells and surrounding mesenchyme, ventricular lining cells in the brain and cartilagenous condensations and surrounding connective tissue in ribs remains at high levels throughout mouse gestation. The expression pattern of CPZ overlaps with the expression pattern of several Wnt genes, consistent with the putative role of CPZ in Wnt signaling.

A cDNA encoding human carboxypeptidase Z (CPZ), a novel metallocarboxypeptidase, was recently cloned (Song and Fricker, J. Biol. Chem., 272, 1054, 1997). In the present study, a cDNA encoding the rat homolog of CPZ was identified. As with the human form, rat CPZ contains an N-terminal domain of 120 amino acids that has 20% to 30% amino acid identity with the "frizzled" domain found on proteins that interact with Wnt, a protein involved in tissue polarity in early embryogenesis. Sequence analysis showed rat and human CPZ to be highly conserved within the frizzled domain (77% amino acid identity), the carboxypeptidase domain (91%), and the C-terminal 28 residues (78%). The entire rat CPZ protein has high sequence similarity with human CPZ (81% amino acid identity), moderate sequence similarity to human carboxypeptidase N (45%), human carboxypeptidase E (41%), and human carboxypeptidase M (33%), and less sequence similarity with other metallocarboxypeptidases. Northern blot analysis showed rat CPZ mRNA to be abundant in the placenta, with low to moderate levels in the brain, lung, thymus, and kidney. The BRL3A rat liver cell line and the PC12 rat adrenal cell line express high levels of CPZ mRNA. In situ hybridization analysis indicated that CPZ is expressed only in specific cell types. For example, in the brain, CPZ mRNA is present in leptomeningeal cells, but not in the majority of other cell types. This distribution in leptomeningeal cells is shared by AEBP1, a recently reported member of the metallocarboxypeptidase gene family. However, the distribution of CPZ and AEBP1 differ in pituitary and thyroid. Taken together, these studies suggest that CPZ functions in a range of cell types.

A novel cDNA, designated carboxypeptidase Z (CPZ), was identified based on its homology to known metallocarboxypeptidases. Northern blot analysis shows bands of 2.1 and/or 2.6 kilobases in all tissues examined. The major form of CPZ mRNA in human salivary gland encodes a protein with an open reading frame of 641 amino acids. In addition, three variants were found that presumably arise due to alternative intron splicing. The 641-amino acid protein contains an 18-residue signal peptide-like sequence, a 120-residue region that shows 23–29% amino acid identity with a Cys-rich domain found in frizzled proteins and in type XVIII collagen, and then a 390-residue carboxypeptidase domain with 49% amino acid identity to carboxypeptidases E and N. The 641-amino acid form of CPZ expressed in the baculovirus system cleaves 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-Phe-Ala-Arg, although the level of enzyme activity was approximately 10-fold lower than either carboxypeptidase E or D expressed using the same viral system. The CPZ activity is more active at neutral pH than at pH 5.5 and is inhibited by active site-directed inhibitors of metallocarboxypeptidases. In summary, CPZ is a novel metallocarboxypeptidase that is active toward substrates with C-terminal basic amino acids.

Background: The biological mechanisms by which cerebral aneurysms emerge, enlarge and rupture are not totally understood. In the present study, we analyzed the genome-wide gene expression profile in human intracranial aneurysms using cDNA microarrays. Methods: Affymetrix HU133 Plus 2.0 microarrays were used to compare gene expression levels between human cerebral aneurismal samples and normal blood vessels. Raw data were evaluated by GeneSpring software version 9.05. Differentially expressed genes were further classified according to the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes annotations. Quantitative real-time polymerase chain reaction was used to confirm the microarray results. Results: Clustering analysis demonstrated that the aneurysm tissues and control tissues were clearly separated into two distinct groups according the differential expression patterns. The levels of 1,160 genes were significantly changed. Of these genes, 164 were upregulated and 996 were downregulated. A cluster of extracellular matrix related genes, including collagens (type I, III, V, and XI) and metalloproteinases were significantly upregulated. In contrast to previous reports, we found that a number of immune/inflammation-related genes were downregulated in intracranial aneurysms. Conclusion: Human cerebral aneurysms appeared to show a similarity in the global gene expression profile. Our results support the notion that disrupted homeostasis of the extracellular matrix components may be involved in the pathogenesis of intracranial aneurysms. Our data of the differentially regulated genes may benefit future studies on the cellular mechanisms of cerebral aneurysms.

Publisher Summary This chapter describes the activity, specificity and structural chemistry of carboxypeptidase Z (CPZ). CPZ is a single-chain protein of approximately 71 kDa. Different splice variants have been detected, with the major form of human CPZ 641 amino acids in length. The crystal structure of CPZ has not been determined, but predictions of the structure have been made based on amino acid sequence similarities to other proteins. As with all members of the metallocarboxypeptidase family, CPZ contains a 300 residue domain that has approximately 20%–50% amino acid sequence identity with other family members. Following this, carboxypeptidase domain is an 80 residue region that has 40–50% amino acid sequence identity with the corresponding region of metallocarboxypeptidase D; this region of metallocarboxypeptidase D has been found to fold into a β sheet-rich structure that has homology with transthyretin. Based on the amino acid sequence similarity between CPZ and this region of metallocarboxypeptidase D, it is likely that the corresponding domain on CPZ also folds into a transthyretin-like domain.Upstream of the carboxypeptidase domain is a 120 residue Cys-rich region that has approximately 20%–30% amino acid sequence identity with a Cys-rich domain present in a number of proteins that bind Wnts.

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Carboxypeptidases may play important role(s) in prohormone processing in normal and neoplastic adenohypophyseal cells of the pituitary. We have recently demonstrated carboxypeptidase E (CPE) and carboxypeptidase Z (CPZ) in the majority of adenohypophyseal cells with carboxypeptidase D (CPD) immunoreactivity largely confined to adrenocorticotrophs. This study evaluated the expression patterns of CPE, CPD, and CPZ immunoreactivity in 48 pituitary adenomas. Our immunohistochemistry demonstrated extensive intracytoplasmic immunoreactivity for CPE, CPD, and CPZ in adrenocorticotrophic hormone (ACTH)-producing adrenocorticotroph cells, prolactin-producing lactotroph cells, and growth hormone (GH)-producing somatotroph cell adenomas, all of which require carboxypeptide processing of prohormones to produce active endocrine hormones. In contrast to the restricted expression in the normal adenohypophysis, CPD appeared to be widespread in the majority of adenomas, suggesting that CPD levels are increased in adenomas. In luteinizing hormone/follicle-stimulating hormone (LH/FSH)-producing gonadotroph adenomas, which do not require carboxypeptidases to produce gonadotropins, only CPZ immunostaining was demonstrated. In null-cell adenomas, CPE immunoreactivity was detected in the majority of tumors, but CPD and CPZ were identified only in a minority of cases. CPE in these cells may process other peptides critical for pituitary cell function, such as chromogranin A or B. These findings suggest that CPs participate in the functioning of pituitary adenomas.

Carboxypeptidases perform many diverse functions in the body. The well-studied pancreatic enzymes (carboxypeptidases A1, A2 and B) are involved in the digestion of food, whereas a related enzyme (mast-cell carboxypeptidase A) functions in the degradation of other proteins. Several members of the metallocarboxypeptidase gene family (carboxypeptidases D, E, M and N) are more selective enzymes and are thought to play a role in the processing of intercellular peptide messengers. Three other members of the metallocarboxypeptidase gene family do not appear to encode active enzymes; these members have been designated CPX-1, CPX-2 and AEBP1/ACLP. In this review, we focus on the recently discovered carboxypeptidase Z (CPZ). This enzyme removes C-terminal Arg residues from synthetic substrates, as do many of the other members of the gene family. However, CPZ differs from the other enzymes in that CPZ is enriched in the extracellular matrix and is broadly distributed during early embryogenesis. In addition to containing a metallocarboxypeptidase domain, CPZ also contains a Cys-rich domain that has homology to Wnt-binding proteins; Wnts are important signaling molecules during development. Although the exact function of CPZ is not yet known, it is likely that this protein plays a role in development by one of several possible mechanisms.

Carboxypeptidase Z (CPZ) is a newly reported member of the metallocarboxypeptidase gene family, but unlike other members of this family, CPZ contains an N-terminal domain that has amino acid sequence similarity to Wnt-binding proteins. In order to gain insights as to the potential function of CPZ, the intracellular localization of this protein was determined in cell culture and in human tissues. When expressed in the AtT-20 mouse pituitary cell line, CPZ protein is routed to the regulated secretory pathway and secreted upon stimulation. A fraction of the secreted CPZ remains associated with the extracellular matrix. Endogenous CPZ in the PC12 rat pheochromocytoma cell line is also associated with the extracellular matrix. In human placenta, CPZ is present within invasive trophoblasts and in the surrounding extracellular space, indicating an association with extracellular matrix. CPZ is also present in amnion cells, but is not readily apparent in the extracellular matrix of this cell type. A human adenocarcinoma of the colon shows expression of CPZ in the extracellular matrix adjacent to malignant cells. Taken together, CPZ appears to be a component of the extracellular matrix in some cell types, where it may function in the binding of Wnt.

Carboxypeptidase Z (CPZ) is a recently discovered member of the metallocarboxypeptidase gene family that has an N-terminal domain related to the Wnt/wingless binding domain of frizzled receptors and other proteins. To further characterize the enzymatic properties of CPZ, the enzyme was purified using Arg- and heparin-affinity columns. CPZ has a neutral pH optimum, and is inhibited by chelating agents and several divalent cations (Zn2+, Mn2+, Cd2+, Cu2+, Hg2+). Active site-directed inhibitors of several other metallocarboxypeptidases also inhibit CPZ activity with moderate potency. CPZ cleaves substrates with C-terminal Arg residues, preferring peptides with an Ala in the penultimate position. No activity is detected toward substrates with an Ile-Arg or a Pro-Arg sequence. The Km for dansyl-Phe-Ala-Arg and dansyl-Pro-Ala-Arg are both approximately 2 mM. Taken together, these data suggests a selective role for CPZ in the processing of extracellular peptides or proteins.

The receptor tyrosine kinase (RTK) MuSK is part of the receptor complex that initiates neuromuscular junction formation in response to agrin [[1]xReceptor tyrosine kinase specific for the skeletal muscle lineage: expression in embryonic muscle, at the neuromuscular junction, and after injury. Valenzuela, DM, Stitt, TN, DiStefano, PS, Rojas, E, Mattsson, K, Compton, DL et al. Neuron. 1995; 15: 573–584Abstract | Full Text PDF | PubMed | Scopus (296)See all References, [2]xThe receptor tyrosine kinase MuSK is required for neuromuscular junction formation in vivo. DeChiara, TM, Bowen, DC, Valenzuela, DM, Simmons, MV, Poueymirou, WT, Thomas, S et al. Cell. 1996; 85: 501–512Abstract | Full Text | Full Text PDF | PubMed | Scopus (590)See all References, [3]xAgrin acts via a MuSK receptor complex. Glass, DJ, Bowen, DC, Stitt, TN, Radziejewski, C, Bruno, J, Ryan, TE et al. Cell. 1996; 85: 513–523Abstract | Full Text | Full Text PDF | PubMed | Scopus (493)See all References]. MuSK, as well as the related orphan RTK-like proteins Ror1 and Ror2 [4xA novel family of cell surface receptors with tyrosine kinase-like domain. Masiakowski, P and Carroll, RD. J Biol Chem. 1992; 267: 26181–26190PubMedSee all References][4], and their homologs in lower organisms, have an extracellular region composed of varying combinations of immunoglobulin and Kringle domains, but they all also have a domain defined by a pattern of 10 cysteine residues.We have noticed that the spacing of cysteines, and a number of other amino acids, is shared between this domain and a cysteine-rich domain (CRD) that serves as the ligand-binding portion of the Frizzled (Fz) family of seven-pass transmembrane receptors for the Wnt signaling molecules [5xA new member of the frizzled family from Drosophila functions as a Wingless receptor. Bhanot, P, Brink, M, Samos, CH, Hsieh, JC, Wang, Y, Macke, JP et al. Nature. 1996; 382: 225–230Crossref | PubMed | Scopus (990)See all References][5]. As seen in other receptor families, the Wnt-binding domain can also be a part of secreted Frizzled-related proteins (sFRPs) [6xA family of secreted proteins contains homology to the cysteine-rich ligand-binding domain of frizzled receptors. Rattner, A, Hsieh, JC, Smallwood, PM, Gilbert, DJ, Copeland, NG, Jenkins, NA, and Nathans, J. Proc Natl Acad Sci USA. 1997; 94: 2859–2863Crossref | PubMed | Scopus (404)See all References][6].The Fz CRD-like sequences have also been identified in Smoothened (Smo), as well as in the α1 chain of type XVIII collagen (Col18), and carboxypeptidase Z (CPZ). Smo is the signaling partner in the Sonic hedgehog (Shh) receptor system. Smo is inactivated by unoccupied Patched (Ptc) protein. Binding of Shh to Ptc relieves this inhibition. The similarity between Smo and Fz, including the CRD, prompted a suggestion [7xPatching up Hedgehog. Nusse, R. Nature. 1996; 384: 119–120Crossref | PubMed | Scopus (15)See all References][7] that the activity of Smo may in addition be modulated by a Wnt-related ligand, though no support for this possibility has been presented.Alternatively, the 10-cysteine domain found in these diverse proteins may be thought of as a general module for protein–protein interactions, evolving in different cases to bind very different counterparts (just like immunoglobulin domains). A computer alignment of the CRDs from representative proteins (Figure 1Figure 1) shows that the Wnt-binding Fz receptors and sFRPs form a distinct group, well separated from the Smo and MuSK/Ror receptors. Thus, determination of the binding specificity of CRDs in these receptors, as well as in Col18 and CPZ, seems likely to reveal new binding capabilities of the 10-cysteine module.Figure 1(a) Multiple sequence alignment of 10-cysteine domains and (b) a phylogenetic tree showing the clustering relationships used to create the alignment. The sequences were downloaded through NCBI Entrez and processed using the programs in the GCG Wisconsin Package [8xSee all References][8].View Large Image | View Hi-Res Image | Download PowerPoint Slide

Carboxypeptidase Z is a serine carboxypeptidase secreted by Absidia zychae NRIC 1199. The cDNA and genomic DNA carrying the scpZ gene encoding carboxypeptidase Z were cloned and sequenced. The nucleotide sequences of the cDNA (1.4 kb) and the genomic DNA (3.3 kb) were analyzed and the intervening sequences were located by a comparison of the two. It was found that the scpZ gene was interrupted by 11 short introns, 50-75 nucleotides in length. Genomic Southern analysis showed that there was only one scpZ gene in the genome of A. zychae. The gene encoded a putative pre-pro-enzyme composed of 409 amino-acid residues of the mature carboxypeptidase Z (M(r) 45,421) and an additional N-terminal sequence of 51 amino-acid residues. The amino-acid sequence around the active serine residue of carboxypeptidase Z (-G-E-S-Y-G-G-) differed from the consensus (-G-E-S-Y-A-G-) which is conserved in most of the serine carboxypeptidases so far analyzed.

A mutant carboxypeptidase Z from Absidia zychae in which Gly137 was replaced by Ala by site-directed mutagenesis was constructed and expressed in Saccharomyces cerevisiae YPH250. The mutant enzyme hydrolyzed C-terminal Pro-X bonds (X = amino acid) more efficiently than the wild-type enzyme and sequentially released amino acids from the C-termini of oligopeptides.