Aminotransferase activity and bioinformatic analysis of 1-aminocyclopropane-1-carboxylate synthase

The mechanistic fate of pyridoxal phosphate (PLP)-dependent enzymes diverge s after the quinonoid intermediate. 1-Aminocyclopropane-1-carboxylate (ACC) synthase, a member of the alpha family of PLP-dependent enzymes, is optimi zed to direct electrons from the quinonoid intermediate to the gamma -carbo n of its substrate, S-adenosyl-L-methionine (SAM), to yield ACC and 5'-meth ylthioadenosine. The data presented show that this quinonoid may also accep t a proton at C-4' of the cofactor to yield alpha -keto acids and the pyrid oxamine phosphate (PMP) form of the enzyme when other amino acids are prese nted as alternative substrates. Addition of excess pyruvate converts the PM P form of the enzyme back to the PLP form. C-alpha-deprotonation from L-Ala is shown by NMR-monitored solvent exchange to be reversible with a rate th at is less than 25-fold slower than that of deprotonation of SAM. The rate- determining step for transamination follows the formation of the quinonoid intermediate. The rate-determining step for alpha,gamma -elimination from e nzyme-bound SAM is likewise shown to occur after C-alpha-deprotonation, and the quinonoid intermediate accumulates during this reaction. BLAST searche s, sequence alignments, and structural comparisons indicate that ACC syntha ses are evolutionarily related to the aminotransferases. In agreement with previously published reports, an absence of homology was found between the alpha and beta families of the PLP-dependent enzyme superfamily.