Kumarasiri Malika, Zhang Weilie, Shi Qicun, Fisher Jed F, Mobashery Shahriar
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, 46556.
Proteins. 2014 Jul;82(7):1348-58. doi: 10.1002/prot.24501. Epub 2014 Feb 6.
The protonation states of the two active-site lysines (Lys69 and Lys235) of PBP 6 of Escherichia coli were explored to understand the active site chemistry of this enzyme. Each lysine was individually mutated to cysteine, and the resultant two mutant proteins were purified to homogeneity. Each protein was denatured, and its cysteine was chemically modified to produce an S-aminoethylated cysteine (γ-thialysine) residue. Following renaturation, the evaluation of the kinetics of the dd-carboxypeptidase activity of PBP 6 as a function of pH was found consistent with one lysine in its free-base (Lys69) and the other in the protonated state (Lys235) for optimal catalysis. The experimental estimates for their pKa values were compared with the pKa values calculated computationally, using molecular-dynamics simulations and a thermodynamic cycle. Study of the γ-thialysine69 showed that lysine at position 69 influenced the basic limb of catalysis, consistent with the fact that the two lysine side chains are in proximity to each other in the active site. Based on these observations, a reaction sequence for PBP 6 is proposed, wherein protonated Lys235 serves as the electrostatic substrate anchor and Lys69 as the conduit for protons in the course of the acylation and deacylation half-reactions.
为了解大肠杆菌PBP 6的活性位点化学性质，对其两个活性位点赖氨酸（Lys69和Lys235）的质子化状态进行了研究。将每个赖氨酸分别突变为半胱氨酸，并将所得的两种突变蛋白纯化至同质。使每种蛋白变性，其半胱氨酸经化学修饰以产生S-氨乙基化半胱氨酸（γ-硫赖氨酸）残基。复性后，发现PBP 6的dd-羧肽酶活性动力学随pH的变化评估结果与一个赖氨酸处于游离碱状态（Lys69）而另一个处于质子化状态（Lys235）时的最佳催化作用一致。使用分子动力学模拟和热力学循环，将它们的pKa值的实验估计值与计算得出的pKa值进行了比较。对γ-硫赖氨酸69的研究表明，69位的赖氨酸影响催化的碱性分支，这与两个赖氨酸侧链在活性位点彼此靠近的事实一致。基于这些观察结果，提出了PBP 6的反应序列，其中质子化的Lys235作为静电底物锚，而Lys69在酰化和脱酰化半反应过程中作为质子的传导途径。
