Lysine-73 is involved in the acylation and deacylation of β-lactamase
Lysine-73 is involved in the acylation and deacylation of β-lactamase
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DOI：
10.1021/bi992681k
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发表时间：
2000-05-02
期刊：
影响因子：
2.9
通讯作者：
Fink, AL
中科院分区：
文献类型：
--
作者：
Lietz, EJ;Truher, H;Fink, AL
赖氨酸73是A类β-内酰胺酶以及丝氨酸青霉素敏感酶家族的其他成员中的保守活性位点残基;其在催化中的作用仍然存在争议和不确定性。地衣芽孢杆菌β-内酰胺酶中Lys 73突变为丙氨酸导致转换速率（k（cat））和催化效率（k（cat）/K-m）的显著降低，以及几种青霉素和头孢菌素底物钟形pH速率曲线（k（cat）/K-m）中pK（1）向较高pH的非常显著的偏移。pK（1）的增加与谷氨酸166附近赖氨酸的正铵基团的去除一致，酸分支归因于谷氨酸166。k（cat）/K-m vs曲线的碱性分支没有明显偏移，如果该分支反映了野生型酶中Lys 73的电离，则可能已经预期到。与野生型相比，突变体在最佳pH下的k（cat）/K-m对于青霉素下降约200倍，对于头孢菌素下降约10（4）倍，表明青霉素与头孢菌素相比催化机制存在显著差异。用K73 A β-内酰胺酶测定的几种底物观察到爆发动力学，表明潜在的分支途径动力学方案和限速脱酰作用。FTIR分析用于确定酰化或脱酰化是否是限速的。一般而言，酰化是头孢菌素底物的限速步骤，而脱酰化是青霉素底物的限速步骤。结果表明，Lys 73在催化机理的酰化和脱酰化步骤中都起着重要作用。这种突变（K73 A）的影响表明，Lys 73在β-内酰胺酶的催化机制中不起一般碱基的作用。K73 A变异体钟形pH速率曲线的存在表明Lys 73不直接负责此类图中的任一分支。很可能Glu 166和Lys 73在维持最佳静电环境以使完全有效的催化活性发生方面对彼此都是重要的。
Lysine 73 is a conserved active-site residue in the class A beta-lactamases, as well as other members of the serine penicillin-sensitive enzyme family; its role in catalysis remains controversial and uncertain. Mutation of Lys73 to alanine in the beta-lactamase from Bacillus licheniformis resulted in a substantial reduction in both turnover rate (k(cat)) and catalytic efficiency (k(cat)/K-m), and a very significant shift in pK(1) to higher pH in the bell-shaped pH-rate profiles (k(cat)/K-m) for several penicillin and cephalosporin substrates. The increase in pK(1) is consistent with the removal of the positive ammonium group of the lysine from the proximity of Glu166, to which the acid limb has been ascribed. The alkaline limb of the k(cat)/K-m vs profiles is not shifted appreciably, as might have been expected if this limb reflected the ionization of Lys73 in the wild-type enzyme. The k(cat)/K-m at the pH optimum for the mutant was down about 200-fold for penicillins and around 10(4) for cephalosporins, compared to the wild-type, suggesting significant differences in the mechanisms for catalysis of penicillins compared to cephalosporins. Burst kinetics were observed with several substrates assayed with K73A beta-lactamase, indicating an underlying branched-pathway kinetic scheme, and rate-limiting deacylation. FTIR analysis was used to determine whether acylation or deacylation was rate-limiting. In general, acylation was the rate-limiting step for cephalosporin substrates, whereas deacylation was rate-limiting for penicillin substrates. The results indicate that Lys73 plays an important role in both the acylation and deacylation steps of the catalytic mechanism. The effects of this mutation (K73A) indicate that Lys73 does not function as a general base in the catalytic mechanism of beta-lactamase. The existence of bell-shaped pH-rate profiles for the K73A variant suggests that Lys73 is not directly responsible for either limb in such plots. It is likely that both Glu166 and Lys73 are important to each other in terms of maintaining the optimum electrostatic environment for fully efficient catalytic activity to occur.