Structure and Function of Histidinol phosphate Aminitrans ferase

HPAT遵循乒乓球BiBi型动力学机制。PLP形式的HPAT表现出pH依赖性的吸收和CD光谱的变化，在高pH时的最大吸收波长为340 nm，在低pH(pKa=6.6)时的最大吸收波长为420 nm。戊二酸或2-甲基谷氨酸结合的野生型酶的吸收光谱变化表明，米氏络合物和外源乙二胺的席夫碱pKa增加。K214A突变酶在2-甲基谷氨酸存在下的吸收光谱表明，外源乙二胺的本征Schiff碱pKa明显升高。甲胺取代K214A突变酶的pK_a值由6.6提高到10.6。Asn157突变为Ala使pK_a增加到9.2。数据表明，在HPAT中，Schiff碱的应变是降低pKa的主要因素，而在催化过程中，Schiff碱的应变是随后增加pKa的关键因素。这表明Schiff碱应变机制是I亚群氨基转移酶的共同特征。

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.