Natarajan S K, Sierks M R
Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County 21228, USA.
Biochemistry. 1996 Dec 3;35(48):15269-79. doi: 10.1021/bi961355r.
Intermediates in the catalytic mechanism of Aspergillus awamori glucoamylase (GA) were identified by studying pre-steady-state and steady-state kinetics of the wild-type GA/maltose and Trp120 -->Phe GA/maltotriose reactions in H2O and D2O. Pre-steady-state fluorescence signal analysis was carried out to ascertain the relative intrinsic fluorescence of the enzyme intermediates. A three-step minimal pathway for oligosaccharide hydrolysis represented by E + Gx (k1) reversible (k-1) EGX (k2)reversible(k-2) EP (kcat)--> E + P is proposed. The first step, represented by the association constant K1 (k1/k-1), depicts the fast formation of enzyme-substrate complex and is the primary factor in fluorescence quenching. A 2.7-fold increase in K1 with D2O as solvent is observed with both enzymes due to the cumulative effect of deuterium on complex hydrogen bonding at the active site. The second step further quenches the enzyme fluorescence and is identified as the hydrolytic step, forming an enzyme-product complex. Both k2 and k-2 values show similar 2-fold decreases in D2O for both enzymes, consistent with the microscopic reversibility of the hydrolytic reaction. The solvent isotopic effect on the hydrolytic step is likely due to either abstraction of an exchangeable proton from the general acid Glu179 or directed addition of water to the oxocarbonium ion intermediate by the general base Glu400. No significant isotope effect was observed on the steady-state kcat value for wild-type GA with maltose, indicating a ronhydrolytic step as rate-limiting. The third step, a posthydrolytic rate-determining step, is the product release as evident from steady-state kinetics with wild-type and Trp120-->Phe GAs using alpha-D-glucosyl fluoride.
通过研究野生型米曲霉葡萄糖淀粉酶（GA）/麦芽糖和色氨酸120→苯丙氨酸GA/麦芽三糖在H₂O和D₂O中的预稳态和稳态动力学来确定其催化机制的中间体。进行预稳态荧光信号分析以确定酶中间体的相对固有荧光。提出了以E + Gx（k1）可逆（k-1）EGX（k2）可逆（k-2）EP（kcat）→E + P表示的寡糖水解的三步最小途径。第一步，以缔合常数K1（k1/k-1）表示，描述了酶-底物复合物的快速形成，是荧光猝灭的主要因素。由于氘对活性位点复合氢键的累积作用，两种酶在以D₂O为溶剂时K1均增加2.7倍。第二步进一步猝灭酶荧光，被确定为水解步骤，形成酶-产物复合物。两种酶的k2和k-2值在D₂O中均显示出类似的2倍下降，这与水解反应的微观可逆性一致。水解步骤的溶剂同位素效应可能是由于从通用酸Glu179中提取可交换质子，或通用碱Glu400将水定向添加到氧鎓离子中间体上。对于野生型GA与麦芽糖的稳态kcat值未观察到明显的同位素效应，表明非水解步骤是限速步骤。第三步，水解后速率决定步骤，是产物释放，这从使用α-D-葡萄糖基氟化物的野生型和色氨酸120→苯丙氨酸GA的稳态动力学中可以明显看出。
