Transient-state kinetics of the reaction of aspartate aminotransferase with aspartate at low pH reveals dual routes in the enzyme-substrate association process.
In aspartate aminotransferase, the coenzyme pyridoxal 5'-phosphate forms a Schiff base with the epsilon-amino group of Lys258. The pH dependency of the steady-state kinetics of the overall reaction had indirectly suggested that the Schiff-base-unprotonated form of the enzyme (EL) is the active species that binds the monoanionic form of aspartate (SH+), the predominant species of the substrate in solution. In order to obtain direct information on the association process, we carried out transient-phase kinetics of the first half-reaction of the enzyme with aspartate at various pH. The disappearance of EL (lambdamax = 358 nm) was fast and independent of pH, but the disappearance of ELH+ (Schiff-base-protonated form, lambdamax = 430 nm) was slow and dependent on pH. At pH values below 6.8 and low concentrations of aspartate, the results could be interpreted to indicate that EL reacts rapidly with SH+ to form the pyridoxamine 5'-phosphate form of the enzyme (EM), and the reaction of ELH+ proceeds via the route ELH+ right arrow over left arrow EL right arrow over left arrow EM, where the first step was found to be rate limiting from the pH jump/drop study of the enzyme. At higher pH values, the rate of disappearance of ELH+ became larger than expected from the above scheme. This deviation became apparent with increasing pH, and could be excellently explained if we consider that it is due to the reaction of ELH+ with the dianionic form of aspartate (S). Thus, the formation of the Michaelis complex of aspartate aminotransferase and aspartate can proceed via two routes; route A is the association of EL with SH+ to form EL.SH+, which converts intramolecularly to ELH+.S, and route B is the association of ELH+ with S to form ELH+.S directly. ELH+.S is the prerequisite structure for further processing of the substrate by the enzyme. The reactions of EM and oxo acids yielded almost exclusively EL and SH+, and therefore route B does not seem to play an essential role in the overall reactions of the enzyme. Route B, however, may be important in the reaction mechanisms of other pyridoxal 5'-phosphate enzymes which have only the ELH+ form.
Hayashi
H
H
Department of Biochemistry, Osaka Medical College, Takatsuki, Japan.
Kagamiyama
H
H
D001219:Aspartate Aminotransferases / Q000737:chemistry*; D001224:Aspartic Acid / Q000737:chemistry*; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011732:Pyridoxal Phosphate / Q000737:chemistry; D012545:Schiff Bases; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D012545:Schiff Bases; D001224:Aspartic Acid; D011732:Pyridoxal Phosphate; D001219:Aspartate Aminotransferases