Stereospecificity of sodium borohydride reduction of Schiff bases at the active site of aspartate aminotransferase (original) (raw)

Sodium b~ro[~H]hydride treatment of holoaspartate aminotransferase results in the reduction of the Schiff's base formed between pyridoxal phosphate and Lys 258. Treatment of the reduced enzyme with papain followed by acid hydrolysis liberates e-N-[3H]pyridoxyl lysine which is degraded to [3HJpyridoxamine diHCl and stereochemically analyzed with apoaspartate aminotransferase. Sodium b~ro[~HJhydride treatment of active site carbamylated aspartate aminotransferase reconstituted with pyridoxyl phosphate and sodium aspartate results in the trapping of an enzyme. substrate complex through the reduction of the Schiffs base formed between pyridoxal phosphate and aspartate. Active site bound N-[3H]pyridoxyl aspartate is liberated by treatment with papain and degraded to [3H]pyridoxamine diHCl for stereochemical analysis. Borohydride reduction of the holoenzyme occurs from the re face of the pyridoxal phosphate Lys 258 Schiff's base. Similarly, reduction of active site carbamylated enzyme substrate complex occurs from the re face of the pyridoxal phosphate-aspartate Schiff s base. These results indicate that when active site carbamylated enzyme binds substrate to pyridoxal phosphate it does so stereospecifically and without changing the face of the Schiff base that is available for reduction as compared to native enzyme. ~ ~~~ Stereochemical studies of aspartate aminotransferase as well as other pyridoxal phosphate-dependent enzymes have led to a detailed understanding of the role played by pyridoxal phosphate in the mechanism of catalysis by those enzymes. Dunathan (1) suggested that one of the primary catalytic functions of pyridoxal phosphate enzymes is to orient the cofactorsubstrate complex in a reactive conformation. Such an orientation has the breaking and forming bonds orthogonal to the plane of the conjugated n system so as to stabilize the developing charge. Also, the cofactor. substrate complex would be rapidly bound by these enzymes on a single face, thereby forcing all of the breaking and making of bonds to occur on the opposite side. In aspartate aminotransferase, the C-a-H bond is broken and the C-4'-H bond formed through a cis process occurring on the si face of the cofactor substrate complex (Scheme I). Recent studies (2-4) have shown that in at least two other pyridoxal phosphate enzymes, reduction of enzyme-bound Schiff s base intermediates is an effective means of determining the exposed "solvent side" of cofactor. substrate com-* This work was supported by Grants GM 24885 and HL 22265 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.