title: Comparison of the enzymatic and functional properties of three cytosolic carboxypeptidase family members.
abstract: Nna1 (CCP1) defines a subfamily of M14 metallocarboxypeptidases (CCP1-6) and is mutated in pcd (Purkinje cell degeneration) mice. Nna1, CCP4, and CCP6 are involved in the post-translational process of polyglutamylation, where they catalyze the removal of polyglutamate side chains. However, it is unknown whether these three cytosolic carboxypeptidases share identical enzymatic properties and redundant biological functions. We show that like Nna1, purified recombinant CCP4 and CCP6 deglutamylate tubulin, but unlike Nna1, neither rescues Purkinje cell degeneration in pcd mice, indicating that they do not have identical functions. Using biotin-based synthetic substrates, we established that the three enzymes are distinguishable based upon individual preferences for glutamate chain length, the amino acid immediately adjacent to the glutamate chain, and whether their activity is enhanced by nearby acidic amino acids. Nna1 and CCP4 remove the C-terminal glutamate from substrates with two or more glutamates, whereas CCP6 requires four or more glutamates. CCP4 behaves as a promiscuous glutamase, with little preference for chain length or neighboring amino acid composition. Besides glutamate chain length dependence, Nna1 and CCP6 exhibit higher k(cat)/K(m) when substrates contain nearby acidic amino acids. All cytosolic carboxypeptidases exhibit a monoglutamase activity when aspartic acid precedes a single glutamate, which, together with their other individual preferences for flanking amino acids, greatly increases the potential substrates for these enzymes and the biological processes in which they act. Additionally, Nna1 metabolized substrates mimicking the C terminus of tubulin in a way suggesting that the tyrosinated form of tubulin will accumulate in pcd mice.
authors.affiliation: From the Departments of Developmental Neurobiology and.
authors.forename: Hui-Yuan
authors.initials: HY
authors.lastname: Wu
authors.affiliation: From the Departments of Developmental Neurobiology and.
authors.forename: Yongqi
authors.initials: Y
authors.lastname: Rong
authors.affiliation: From the Departments of Developmental Neurobiology and.
authors.forename: Kristen
authors.initials: K
authors.lastname: Correia
authors.affiliation: Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee 38105.
authors.forename: Jaeki
authors.initials: J
authors.lastname: Min
authors.affiliation: From the Departments of Developmental Neurobiology and jim.morgan@stjude.org.
authors.forename: James I
authors.initials: JI
authors.lastname: Morgan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002268:Carboxypeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D009410:Nerve Degeneration / Q000378:metabolism* / Q000473:pathology; D011099:Polyglutamic Acid / Q000378:metabolism*; D011689:Purkinje Cells / Q000378:metabolism / Q000473:pathology; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity; D014404:Tubulin / Q000378:metabolism
chemical_list: D014404:Tubulin; D011099:Polyglutamic Acid; C000596978:CCP4 protein, mouse; C000596944:CCP6 protein, mouse; D002268:Carboxypeptidases; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; C420042:Agtpbp1 protein, mouse; D019204:GTP-Binding Proteins
keywords: Cerebellum; Cytosolic Carboxypeptidase; Enzyme Kinetics; Neurodegeneration; Polyglutamylation; Post-translational Modification (PTM); Transgenic Mice