GO:0004185 serine-type carboxypeptidase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0004185 serine-type carboxypeptidase activity describes the hydrolysis of a single C-terminal amino acid from a polypeptide chain using a catalytic triad of serine, histidine and aspartate/glutamate.
• Serine-type carboxypeptidases are widespread in fungi, where they process propeptides and regulate cell wall remodelling and hyphal growth.
• The catalytic mechanism involves a nucleophilic serine activated by a proton relay, forming an acyl-enzyme intermediate that is resolved by water.
• Aspergillus oryzae serine-type carboxypeptidases such as OcpA, OcpB, OcpC and KexA show distinct substrate specificities and expression patterns.
• Dysregulation of serine-type carboxypeptidase activity can affect fungal virulence, protein maturation and industrial enzyme production.
• CRISPR knockout, point mutation, knock-in and overexpression models enable causal dissection of serine-type carboxypeptidase genes in fungal and human cells.
Description
Serine-type carboxypeptidase activity (GO:0004185) is a molecular function that removes a single amino acid from the C-terminus of a polypeptide chain using a catalytic triad with a serine nucleophile. This activity is essential for protein maturation, peptide hormone processing and cell wall remodelling in fungi and other organisms. Researchers study GO:0004185 to understand how proteolytic processing controls development, virulence and industrial enzyme production. The QuickGO definition specifies that catalysis proceeds via a serine nucleophile activated by a proton relay involving an acidic residue (aspartate or glutamate) and a basic residue (usually histidine). In Aspergillus oryzae, multiple serine-type carboxypeptidases such as OcpA, OcpB, OcpC and KexA have been characterized, revealing diversity in substrate specificity and regulation. These enzymes are also relevant to biotechnology because they influence the yield and quality of secreted proteins.
serine-type carboxypeptidase activity At A Glance
| GO ID | GO:0004185 |
|---|---|
| GO term | serine-type carboxypeptidase activity |
| Ontology | molecular_function |
| Synonym | serine carboxypeptidase activity |
| Definition | Catalysis of the hydrolysis of a single C-terminal amino acid residue from the C-terminus of a polypeptide chain by a catalytic mechanism that involves a catalytic triad consisting of a serine nucleophile that is activated by a proton relay involving an acidic residue (e.g. aspartate or glutamate) and a basic residue (usually histidine). |
| Major function | C-terminal proteolytic processing of peptides and proteins |
| Catalytic residues | Serine nucleophile, histidine base, aspartate/glutamate acid |
| Representative genes | ocpG, CpI, OcpA, OcpB, OcpC, KexA |
| Organisms studied | Aspergillus oryzae, Saccharomyces cerevisiae, other fungi |
What Is GO:0004185?
GO:0004185 serine-type carboxypeptidase activity is defined as catalysis of the hydrolysis of a single C-terminal amino acid residue from the C-terminus of a polypeptide chain by a catalytic mechanism that involves a catalytic triad consisting of a serine nucleophile that is activated by a proton relay involving an acidic residue (e.g. aspartate or glutamate) and a basic residue (usually histidine). In simpler terms, it is a serine protease that trims one amino acid at a time from the end of a protein or peptide.
Why Is serine-type carboxypeptidase activity Important in Cell Biology?
Serine-type carboxypeptidase activity is important because it controls the final step of proteolytic processing for many proteins and peptides, influencing fungal development, cell wall integrity and secretion. In Aspergillus oryzae, these enzymes are required for normal hyphal growth and conidiation, and their substrate specificity differs from that of Saccharomyces cerevisiae Kex1. Understanding GO:0004185 helps researchers engineer industrial strains for better protein production and to study fungal pathogenesis.
• Controls C-terminal trimming of proteins and peptides, affecting their activity and stability.
• Required for normal hyphal growth and conidiation in Aspergillus oryzae.
• Contributes to cell wall remodelling and protein secretion in fungi.
• Shows species-specific substrate specificity, as seen for KexA versus Kex1.
• Influences industrial enzyme production by Aspergillus species.
• Provides a model for studying serine protease catalytic triads.
• Relevant to antifungal target discovery due to roles in fungal growth.
• Enables biotechnological processing of peptides and proteins.
• Linked to mRNA processing through intron retention of ocpG.
• Useful for comparative enzymology across fungal species.
Molecular Mechanism of serine-type carboxypeptidase activity
Substrate recognition and binding
In simple terms: The enzyme grabs the end of a protein chain.
Serine-type carboxypeptidases bind the C-terminus of a polypeptide chain in a substrate-binding cleft that positions the terminal peptide bond for cleavage. The enzyme recognizes the free carboxylate group of the C-terminal residue, which anchors the substrate in the active site.
Catalytic triad activation
In simple terms: Three amino acids work together to make serine reactive.
The catalytic triad consists of a serine nucleophile, a histidine base and an acidic residue (aspartate or glutamate). The histidine abstracts a proton from the serine hydroxyl, increasing its nucleophilicity, while the acidic residue stabilizes the histidine positive charge.
Acyl-enzyme intermediate formation
In simple terms: The enzyme temporarily holds onto part of the protein.
The activated serine attacks the carbonyl carbon of the C-terminal peptide bond, forming a covalent acyl-enzyme intermediate and releasing the C-terminal amino acid. This step is characteristic of serine proteases and distinguishes them from metallocarboxypeptidases.
Deacylation and product release
In simple terms: Water comes in to finish the job and release the trimmed protein.
A water molecule, activated by the histidine, hydrolyzes the acyl-enzyme intermediate, releasing the truncated polypeptide and regenerating the free enzyme. The enzyme is then ready for another round of catalysis.
Regulation by expression and intron retention
In simple terms: Cells control how much enzyme is made.
In Aspergillus oryzae, mRNA expression of the serine-type carboxypeptidase ocpG is regulated through intron retention, producing different transcripts. This post-transcriptional mechanism adds a layer of control over enzyme levels.
Key Genes Involved in GO:0004185 serine-type carboxypeptidase activity
The following genes and proteins are experimentally characterized members or regulators of serine-type carboxypeptidase activity (GO:0004185).
| Gene | Major Role | Research Relevance |
|---|---|---|
| ocpG | Serine-type carboxypeptidase in Aspergillus oryzae | mRNA expression regulated by intron retention |
| CpI | Serine-type carboxypeptidase from Aspergillus oryzae | Heterologous expression and characterization |
| OcpA | Serine-type carboxypeptidase from Aspergillus oryzae | Heterologous expression and characterization |
| OcpB | Novel serine-type carboxypeptidase from Aspergillus oryzae | Heterologous expression and characterization |
| OcpC | Unique serine-type carboxypeptidase in Aspergillus oryzae | Enzymatic properties characterized |
| KexA | Serine-type carboxypeptidase in Aspergillus oryzae | Broader substrate specificity than Kex1; required for hyphal growth and conidiation |
| Kex1 | Serine-type carboxypeptidase in Saccharomyces cerevisiae | Reference for substrate specificity comparison |
| PBP1b | Penicillin-binding protein in bacteria | Allosteric activation by LpoB; not a serine carboxypeptidase but related to serine-active-site enzymes |
| PBP1a | Penicillin-binding protein in Streptococcus pneumoniae | Activated by S protein; related to peptidoglycan remodelling |
| β-lactamases | Serine enzymes that hydrolyze β-lactams | Mechanistic parallels to serine proteases |
| LpoB | Outer membrane lipoprotein | Transiently binds and activates PBP1b |
| S protein | Streptococcus pneumoniae protein | Activates PBP1a to regulate peptidoglycan remodelling |
| Glycopeptides | Substrates for carboxypeptidase processing | Clinical relevance of glycopeptide antibiotics |
How Is serine-type carboxypeptidase activity Regulated?
Serine-type carboxypeptidase activity is regulated at multiple levels. In Aspergillus oryzae, ocpG mRNA expression is controlled by intron retention, which alters the amount of functional transcript. The enzyme KexA is required for normal hyphal growth and conidiation, indicating developmental regulation. Substrate specificity differences between KexA and Saccharomyces cerevisiae Kex1 suggest species-specific regulatory roles. Additionally, heterologous expression studies show that OcpA, OcpB and OcpC have distinct enzymatic properties, implying differential regulation of their activities.
serine-type carboxypeptidase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| KexA | Fungal growth and conidiation defects | Aspergillus oryzae knockout and point mutation |
| ocpG | mRNA processing and enzyme expression | Intron retention reporter assays |
| OcpA | Protein secretion and processing | Heterologous expression in Aspergillus |
| OcpB | Novel carboxypeptidase function | Knockout and overexpression in Aspergillus |
| OcpC | Unique enzymatic properties | Recombinant enzyme characterization |
Fungal pathogenesis and virulence
Serine-type carboxypeptidases contribute to fungal growth and cell wall remodelling, processes that are critical for virulence in pathogenic fungi. KexA is required for normal hyphal growth and conidiation in Aspergillus oryzae, and its loss impairs development. Because similar enzymes exist in human pathogens, they are potential antifungal targets.
Industrial biotechnology and protein production
In Aspergillus oryzae, serine-type carboxypeptidases such as OcpA, OcpB and OcpC influence the processing of secreted proteins, affecting yields of industrial enzymes. Understanding their activity helps optimize heterologous protein production.
Glycopeptide processing and antibiotic research
Glycopeptides are substrates for carboxypeptidase-like processing, and their clinical use is relevant to understanding protease specificity. Although the cited work focuses on glycopeptide antibiotics, it highlights the broader importance of C-terminal processing in drug development.
From serine-type carboxypeptidase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Is KexA required for hyphal growth? | KexA knockout in Aspergillus oryzae |
| Does intron retention regulate ocpG expression? | ocpG minigene with retained intron |
| What is the substrate specificity of OcpC? | Recombinant OcpC overexpression and enzyme assays |
| Can OcpA and OcpB be produced heterologously? | Heterologous expression in Aspergillus |
| Does a catalytic serine mutation abolish activity? | Point mutation of serine nucleophile |
| Can KexA complement Kex1 function? | Knock-in of KexA into Saccharomyces kex1 mutants |
How to Study the serine-type carboxypeptidase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Chromogenic peptide assay | Carboxypeptidase activity | Kinetic characterization of OcpA, OcpB, OcpC |
| Heterologous expression | Recombinant enzyme production | Purification of CpI, OcpA, OcpB |
| RT-PCR | mRNA levels and splice variants | ocpG intron retention analysis |
| RNA-seq | Transcriptome-wide expression | Identification of carboxypeptidase genes |
| Gene knockout | Loss-of-function phenotype | KexA growth and conidiation defects |
| Site-directed mutagenesis | Catalytic residue function | Serine nucleophile mutation |
| Enzyme kinetics | Substrate specificity and rate | Comparison of KexA and Kex1 |
| Phylogenetic analysis | Evolutionary relationships | Family classification of serine carboxypeptidases |
Enzyme activity assays
Serine-type carboxypeptidase activity is measured using synthetic peptide substrates with a C-terminal reporter group, such as chromogenic or fluorogenic amino acids. These assays determine kinetic parameters and substrate specificity.
Heterologous expression and purification
Genes such as CpI, OcpA, OcpB and OcpC are expressed in heterologous hosts, and the recombinant enzymes are purified for biochemical characterization. This approach allows comparison of enzymatic properties across family members.
mRNA expression analysis
Transcript levels of serine-type carboxypeptidase genes are quantified by RT-PCR or RNA-seq, revealing regulation by intron retention as shown for ocpG. This method identifies splice variants and expression patterns.
Phenotypic analysis of mutants
Knockout or knockdown strains are examined for growth, conidiation and cell wall integrity, as demonstrated for KexA. Phenotypic screens link enzyme activity to developmental processes.
How CRISPR Can Be Used to Study GO:0004185 serine-type carboxypeptidase activity
Knockout
CRISPR knockout of serine-type carboxypeptidase genes such as KexA in Aspergillus oryzae can reveal loss-of-function phenotypes, including defects in hyphal growth and conidiation. Knockout studies help determine whether a candidate gene is required for normal development.
Point Mutation
Point mutations in the catalytic triad residues (serine, histidine, aspartate/glutamate) can abolish enzyme activity, as inferred from the mechanism. CRISPR point mutation allows testing of specific residues without deleting the entire gene.
Knock-in
Knock-in of epitope tags or fluorescent proteins at the endogenous locus enables visualization and purification of serine-type carboxypeptidases. This approach preserves native regulation and can be used to study localization.
Overexpression
CRISPR activation or overexpression constructs can increase serine-type carboxypeptidase levels, facilitating biochemical characterization and industrial enzyme production. Overexpression of OcpC, for example, allows detailed enzymatic analysis.
How EDITGENE Supports serine-type carboxypeptidase activity Research
Researchers studying serine-type carboxypeptidase activity-related genes often need to determine whether a candidate gene is causally involved in protein processing, fungal development or industrial enzyme production. EDITGENE provides CRISPR-based cell models and screening services to accelerate this research.
Contact EDITGENE today to design your custom CRISPR model for serine-type carboxypeptidase activity research.
Related Products
| Product name | Cat.No. | Species | Gene ID | |
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| CPD Knockout HEK293 Cell Line | EDJ-KQ2097 | Human | 1362 | Details Get a Quote |
| CTSA Knockout HEK293 Cell Line | EDJ-KQ5514 | Human | 5476 | Details Get a Quote |
| PRCP Knockout HEK293 Cell Line | EDJ-KQ5527 | Human | 5547 | Details Get a Quote |
| CPVL Knockout HEK293 Cell Line | EDJ-KQ11443 | Human | 54504 | Details Get a Quote |
| SCPEP1 Knockout HEK293 Cell Line | EDJ-KQ15192 | Human | 59342 | Details Get a Quote |
| CPD Knockout A-549 Cell Line | EDJ-KQ22199 | Human | 1362 | Details Get a Quote |
| CPD Knockout HCT 116 Cell Line | EDJ-KQ22200 | Human | 1362 | Details Get a Quote |
| CPD Knockout HeLa Cell Line | EDJ-KQ22201 | Human | 1362 | Details Get a Quote |
| SCPEP1 Knockout A-549 Cell Line | EDJ-KQ47989 | Human | 59342 | Details Get a Quote |
| SCPEP1 Knockout HCT 116 Cell Line | EDJ-KQ47991 | Human | 59342 | Details Get a Quote |
| SCPEP1 Knockout HeLa Cell Line | EDJ-KQ47992 | Human | 59342 | Details Get a Quote |
| CTSA Knockout A-549 Cell Line | EDJ-KQ27509 | Human | 5476 | Details Get a Quote |
| PRCP Knockout HCT 116 Cell Line | EDJ-KQ27523 | Human | 5547 | Details Get a Quote |
| CTSA Knockout HCT 116 Cell Line | EDJ-KQ28758 | Human | 5476 | Details Get a Quote |
| CTSA Knockout HeLa Cell Line | EDJ-KQ28759 | Human | 5476 | Details Get a Quote |
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Frequently Asked Questions About serine-type carboxypeptidase activity
What is serine-type carboxypeptidase activity?
It is a molecular function (GO:0004185) that removes a single C-terminal amino acid from a polypeptide using a serine catalytic triad.
What genes are involved in serine-type carboxypeptidase activity?
Genes include ocpG, CpI, OcpA, OcpB, OcpC and KexA in Aspergillus oryzae, and Kex1 in Saccharomyces cerevisiae.
What is the catalytic mechanism of serine-type carboxypeptidases?
A serine nucleophile activated by a histidine-aspartate/glutamate proton relay forms an acyl-enzyme intermediate that is hydrolyzed by water.
How is serine-type carboxypeptidase activity regulated?
It is regulated at the mRNA level by intron retention, as shown for ocpG, and by developmental cues.
What diseases are linked to serine-type carboxypeptidase activity?
Dysregulation affects fungal growth and virulence, and these enzymes are potential antifungal targets.
What methods study serine-type carboxypeptidase activity?
Enzyme assays, heterologous expression, RT-PCR, RNA-seq and mutant phenotyping are commonly used.
Can CRISPR knockout be used to study serine-type carboxypeptidases?
Yes, knockout of KexA in Aspergillus oryzae revealed growth and conidiation defects.
What is the difference between serine-type and metallocarboxypeptidases?
Serine-type uses a serine catalytic triad, while metallocarboxypeptidases use a metal ion for catalysis.
Which organisms have serine-type carboxypeptidases?
They are found in fungi such as Aspergillus oryzae and Saccharomyces cerevisiae, and in other eukaryotes.
How can I model serine-type carboxypeptidase mutations?
EDITGENE offers CRISPR knockout, point mutation, knock-in and overexpression models for these genes.
Conclusion
Serine-type carboxypeptidase activity (GO:0004185) is a fundamental proteolytic function that trims C-terminal amino acids using a serine catalytic triad. Its roles in fungal growth, protein processing and industrial enzyme production make it a valuable target for both basic and applied research. CRISPR-based models from EDITGENE enable precise dissection of these enzymes in relevant cell systems.
References
- 1. Léone M et al.. 2000. [Glycopeptides].. Ann Fr Anesth Reanim 19(3):177-87 PMID: 10782241
- 2. Shlosman I et al.. 2025. The hit-and-run of cell wall synthesis: LpoB transiently binds and activates PBP1b through a conserved allosteric switch.. Nat Commun 16(1):6723 PMID: 40691462
- 3. Pratt RF. 2016. β-Lactamases: Why and How.. J Med Chem 59(18):8207-20 PMID: 27232275
- 4. Millat H et al.. 2026. Streptococcus pneumoniae S protein activates PBP1a to regulate peptidoglycan remodelling and cell division.. Nat Microbiol 11(1):301-316 PMID: 41420061
- 5. Ishida K et al.. 2014. Diversity in mRNA expression of the serine-type carboxypeptidase ocpG in Aspergillus oryzae through intron retention.. Biosci Biotechnol Biochem 78(8):1328-36 PMID: 25130734
- 6. Morita H et al.. 2009. Heterologous expression and characterization of CpI, OcpA, and novel serine-type carboxypeptidase OcpB from Aspergillus oryzae.. Appl Microbiol Biotechnol 85(2):335-46 PMID: 19557408
- 7. Morita H et al.. 2011. Enzymatic properties of the recombinant serine-type carboxypeptidase OcpC, which is unique to Aspergillus oryzae.. Biosci Biotechnol Biochem 75(4):662-8 PMID: 21512241
- 8. Morita H et al.. 2012. Serine-type carboxypeptidase KexA of Aspergillus oryzae has broader substrate specificity than Saccharomyces cerevisiae Kex1 and is required for normal hyphal growth and conidiation.. Appl Environ Microbiol 78(22):8154-7 PMID: 22961905
