Structure and Function of Histidinol phosphate Aminitrans ferase

HPAT反应遵循乒乓式动力学机制。PLP型HPAT具有pH依赖性的吸收和CD光谱变化，在高pH时在340 nm处有最大吸收，在低pH时在420 nm处有最大吸收（pK_a=6.6）。与谷氨酸或2-甲基谷氨酸结合的野生型酶的吸收光谱的变化表明，米氏络合物和外部醛亚胺的Schiff碱pK_a增加。K214 A突变酶在2-甲基谷氨酸存在下的吸收光谱表明，外源醛亚胺的固有Schiff碱pK_a明显升高。K214 A突变体酶的pK_a值从6.6增加到10.6。Asn 157突变为Ala使pK_a增加到9.2。实验结果表明，Schiff碱的应变是导致HPAT中pK_a降低的主要因素，对催化过程中Schiff碱pK_a的升高起着决定性的作用。这表明Schiff碱应变机制是亚群Ⅰ氨基转移酶的共同机制。

HPAT followed a Ping-Pong Bi Bi kinetic mechanism. The PLP-form HPAT showed pH dependent absorption and CD spectral changes with maxima at 340 nm at high pH and 420 nm at loW pH (pK_a=6.6). Absorption spectral changes of the glutarate-or 2-methylglutamate-bound wild type enzyme indicated that the Schiff base pK_a of the Michaelis complex and the external aldimine were increased. Absorption spectra of K214A mutant enzyme in the presence of 2-methylglutamate demonstrated that the intrinsic Schiff base pK_a of the external aldimine was much elevated. The pK_a value of the methylamine-substituted K214A mutant enzyme was increased from 6.6 to 10.6. Mutation of Asn157 to Ala increased the pK_a to 9.2. Data indicate that the strain of the Schiff base is the principal factor to decrease the pK_a in HPAT, and is crucial for the subsequent increase in the Schiff base pK_a during catalysis. It is suggested that the Schiff base strain mechanism is a common to the Subgroup I aminotransferases.