Modulation of the internal aldimine pK(a)'s of 1-aminocyclopropane-1-carboxylate synthase and aspartate aminotransferase by specific active site residues.
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Eliot AC, Kirsch JF
Modulation of the internal aldimine pK(a)'s of 1-aminocyclopropane-1-carboxylate synthase and aspartate aminotransferase by specific active site residues.
Biochemistry. 2002 Mar 19;41(11):3836-42.
- PubMed ID
- 11888303 [ View in PubMed]
- Abstract
The active sites of the homologous pyridoxal phosphate- (PLP-) dependent enzymes 1-aminocyclopropane-1-carboxylate (ACC) synthase and aspartate aminotransferase (AATase) are almost entirely conserved, yet the pK(a)'s of the two internal aldimines are 9.3 and 7.0, respectively, to complement the substrate pK(a)'s (S-adenosylmethionine pK(a) = 7.8 and aspartate pK(a) = 9.9). This complementation is required for maximum enzymatic activity in the physiological pH range. The most prominent structural difference in the active site is that Ile232 of ACC synthase is replaced by alanine in AATase. The I232A mutation was introduced into ACC synthase with a resulting 1.1 unit decrease (from 9.3 to 8.2) in the aldimine pK(a), thus identifying Ile232 as a major determinant of the high pK(a) of ACC synthase. The mutation also resulted in reduced k(cat) (0.5 vs 11 s(-1)) and k(cat)/K(m) values (5.0 x 10(4) vs 1.2 x 10(6) M(-1) s(-1)). The effect of the mutation is interpreted as the result of shortening of the Tyr233-PLP hydrogen bond. Addition of the Y233F mutation to the I232A ACC synthase to generate the double mutant I232A/Y233F raised the pK(a) from 8.2 to 8.8, because the Y233F mutation eliminates the hydrogen bond between that residue and PLP. The introduction of the retro mutation A224I into AATase raised the aldimine pK(a) of that enzyme from 6.96 to 7.16 and resulted in a decrease in single-turnover k(max) (108 vs 900 s(-1) for aspartate) and k(max)/K(m)(app) (7.5 x 10(4) vs 3.8 x 10(5) M(-1) s(-1)) values. The distance from the pyridine nitrogen of the cofactor to a conserved aspartate residue is 2.6 A in AATase and 3.8 A in ACC synthase. The D230E mutation introduced into ACC synthase to close this distance increases the aldimine pK(a) from 9.3 to 10.0, as would be predicted from a shortened hydrogen bond.
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- Drug Targets
Drug Target Kind Organism Pharmacological Action Actions Pyridoxal phosphate Aspartate aminotransferase, cytoplasmic Protein Humans UnknownActivatorDetails