Richard J P
Biochemistry. 1985 Feb 12;24(4):949-53. doi: 10.1021/bi00325a021.
Triosephosphate isomerase catalyzes the isomerization and/or racemization reactions of L-glyceraldehyde 3-phosphate (LGAP), the enantiomer of the physiological substrate. The reaction is inhibited by the active site directed reagent glycidol phosphate. The amount of protonation product formation catalyzed by a fixed enzyme concentration is nearly independent of increasing steady-state concentrations of triose 1,2-enediol 3-phosphate caused by buffer catalysis of LGAP deprotonation. Therefore, enzymatic protonation of the enediol or enediolate, which could account for the observed enzymatic catalysis of LGAP isomerization and/or racemization, is at best a minor reaction. Instead LGAP reacts directly at the enzyme active site. Triosephosphate isomerase catalysis of the protonation of triose 1,2-enediol 3-phosphate was expected because of the strong evidence supporting an enediol reaction intermediate for the overall reaction catalyzed by isomerase. The most reasonable explanation for the failure to observe enzymatic protonation is that in solution the enediol undergoes beta elimination of phosphate (t 1/2 is estimated to be 10(-6) s) faster than it can diffuse to and form a complex with isomerase.
磷酸丙糖异构酶催化L-甘油醛-3-磷酸（LGAP）的异构化和/或消旋反应，LGAP是生理底物的对映体。该反应受到活性位点导向试剂磷酸缩水甘油的抑制。在固定酶浓度催化下形成的质子化产物量几乎与因LGAP去质子化的缓冲催化而导致的1,2-烯二醇-3-磷酸丙糖稳态浓度增加无关。因此，烯二醇或烯二醇盐的酶促质子化，虽然可以解释所观察到的LGAP异构化和/或消旋反应的酶促催化作用，但充其量只是一个次要反应。相反，LGAP直接在酶活性位点发生反应。由于有强有力的证据支持异构酶催化的整体反应存在烯二醇反应中间体，所以预计磷酸丙糖异构酶会催化1,2-烯二醇-3-磷酸丙糖的质子化。未能观察到酶促质子化的最合理原因是，在溶液中烯二醇经历磷酸的β消除反应（半衰期估计为10⁻⁶秒）的速度比它扩散到异构酶并与其形成复合物的速度更快。
