Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461.
Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721.
Proc Natl Acad Sci U S A. 2021 Oct 5;118(40). doi: 10.1073/pnas.2109118118.
Heavy enzyme isotope effects occur in proteins substituted with H-, C-, and N-enriched amino acids. Mass alterations perturb femtosecond protein motions and have been used to study the linkage between fast motions and transition-state barrier crossing. Heavy enzymes typically show slower rates for their chemical steps. Heavy bacterial methylthioadenosine nucleosidases (MTANs from and ) gave normal isotope effects in steady-state kinetics, with slower rates for the heavy enzymes. However, both enzymes revealed rare inverse isotope effects on their chemical steps, with faster chemical steps in the heavy enzymes. Computational transition-path sampling studies of and MTANs indicated closer enzyme-reactant interactions in the heavy MTANs at times near the transition state, resulting in an improved reaction coordinate geometry. Specific catalytic interactions more favorable for heavy MTANs include improved contacts to the catalytic water nucleophile and to the adenine leaving group. Heavy bacterial MTANs depart from other heavy enzymes as slowed vibrational modes from the heavy isotope substitution caused improved barrier-crossing efficiency. Improved sampling frequency and reactant coordinate distances are highlighted as key factors in MTAN transition-state stabilization.
重同位素酶效应发生在被 H、C 和 N 富集氨基酸取代的蛋白质中。质量的改变会扰乱皮秒级别的蛋白质运动，并被用于研究快速运动与过渡态势垒穿越之间的联系。重酶通常表现出较慢的化学反应速率。重细菌甲基硫腺苷核苷酶（来自 和 ）在稳态动力学中表现出正常的同位素效应，重酶的反应速率较慢。然而，两种酶在其化学反应步骤中都表现出罕见的逆同位素效应，重酶的化学反应更快。对 和 甲基硫腺苷核苷酶的计算过渡态抽样研究表明，在过渡态附近的时间点，重甲基硫腺苷核苷酶与酶-反应物的相互作用更紧密，从而改善了反应坐标的几何形状。对重甲基硫腺苷核苷酶更有利的特定催化相互作用包括与催化水分子亲核试剂和腺嘌呤离去基团的接触得到改善。重细菌甲基硫腺苷核苷酶与其他重酶不同，因为重同位素取代引起的振动模式减缓提高了势垒穿越效率。提高抽样频率和反应物坐标距离被强调为 MTAN 过渡态稳定的关键因素。
