Comparison of the Enzymatic and Functional Properties of Three Cytosolic Carboxypeptidase Family Members [Neurobiology]

November 21st, 2014 by Wu, H.-Y., Rong, Y., Correia, K., Min, J., Morgan, J. I.

Nna1 (a.k.a. CCP1) defines a subfamily of M14 metallo-carboxypeptidases (CCP1-6) and is mutated in Purkinje cell degeneration (pcd) mice. Nna1, CCP4 and CCP6 are involved in the posttranslational process of polyglutamylation, where they catalyze the removal of polyglutamate side-chains. However it is unknown whether these three CCPs share identical enzymatic properties and redundant biological functions. We show that like Nna1, purified recombinant CCP4 and CCP6 deglutamylate tubulin, but unlike Nna1, neither rescue Purkinje cell degeneration in pcd mice, indicating they do not have identical functions. Using biotin-based synthetic substrates, we establish 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 2 or more glutamates whereas CCP6 requires 4 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 kcat/KM when substrates contained nearby acidic amino acids. All CCPs exhibit a monoglutamase activity when aspartic acid precedes a single glutamate, which together with their other individual preferences for flanking amino acids greatly increase 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 the tyrosinated form of tubulin will accumulate in pcd mice.