Hayashi H, Kagamiyama H
Department of Biochemistry, Osaka Medical College, Takatsuki, Japan.
Biochemistry. 1997 Nov 4;36(44):13558-69. doi: 10.1021/bi971638z.
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.
在天冬氨酸转氨酶中，辅酶磷酸吡哆醛与赖氨酸258的ε-氨基形成席夫碱。总反应稳态动力学的pH依赖性间接表明，酶的席夫碱未质子化形式（EL）是结合天冬氨酸单阴离子形式（SH⁺）的活性物种，SH⁺是溶液中底物的主要存在形式。为了获得有关缔合过程的直接信息，我们在不同pH值下对酶与天冬氨酸的第一个半反应进行了瞬态动力学研究。EL（λmax = 358 nm）的消失很快且与pH无关，但ELH⁺（席夫碱质子化形式，λmax = 430 nm）的消失很慢且与pH有关。在pH值低于6.8且天冬氨酸浓度较低时，结果可以解释为表明EL与SH⁺快速反应形成酶的磷酸吡哆胺5'-磷酸形式（EM），ELH⁺的反应通过ELH⁺⇌EL⇌EM途径进行，其中第一步从酶的pH跃变/下降研究中发现是限速步骤。在较高pH值下，ELH⁺的消失速率变得比上述方案预期的要大。随着pH升高，这种偏差变得明显，如果我们认为这是由于ELH⁺与天冬氨酸的双阴离子形式（S）反应所致，那么就可以很好地解释这种偏差。因此，天冬氨酸转氨酶与天冬氨酸的米氏复合物的形成可以通过两条途径进行；途径A是EL与SH⁺缔合形成EL.SH⁺，其分子内转化为ELH⁺.S，途径B是ELH⁺与S缔合直接形成ELH⁺.S。ELH⁺.S是酶进一步处理底物的前提结构。EM与氧代酸的反应几乎只产生EL和SH⁺，因此途径B似乎在酶的整体反应中不发挥重要作用。然而，途径B可能在其他仅具有ELH⁺形式的磷酸吡哆醛5'-磷酸酶的反应机制中很重要。
