Monnaie D, Dubus A, Frère J M
Centre d'ingénierie des Protéines and Laboratoire d'Enzymologie, Université de Liège, Belgium.
Biochem J. 1994 Aug 15;302 ( Pt 1)(Pt 1):1-4. doi: 10.1042/bj3020001.
By using site-directed mutagenesis, the conserved Lys-67 residue situated three positions after the active-site Ser of a class C beta-lactamase was replaced by Arg or Gln. The Lys-67-Gln protein was nearly inactive. Although severely impaired, the Lys-67-Arg mutant exhibited an appreciable activity above pH 7.5 and, for some poor substrates of the wild-type enzyme, the kcat. values were even increased. The properties of the Lys-67-Arg mutant were studied by both steady-state and transient-state kinetic methods with a variety of compounds representing distinct classes of available substrates. With beta-lactam substrates, the kcat./Km values reflecting the efficiency of the acylation step (k+2/K) were decreased 25-100-fold. When the individual values could be measured, k+2 was not significantly altered, but K was found to be strongly increased, a result most likely explained by a corresponding increase in the k+1/k-1 ratio. These results, combined with the much stronger impairment of the Lys-67-Gln mutant, can be interpreted by attributing an electrostatic role to the positive ammonium group of the Lys-67 side chain.
通过定点诱变，将C类β-内酰胺酶活性位点丝氨酸后三个位置的保守赖氨酸-67残基替换为精氨酸或谷氨酰胺。赖氨酸-67-谷氨酰胺蛋白几乎没有活性。尽管严重受损，但赖氨酸-67-精氨酸突变体在pH 7.5以上仍表现出相当的活性，并且对于野生型酶的一些劣质底物，催化常数（kcat）值甚至有所增加。采用稳态和瞬态动力学方法，用代表不同类型可用底物的多种化合物研究了赖氨酸-67-精氨酸突变体的性质。对于β-内酰胺底物，反映酰化步骤效率（k+2/K）的kcat/Km值降低了25至100倍。当可以测量各个值时，k+2没有显著变化，但发现K大幅增加，这一结果很可能是由k+1/k-1比值相应增加所解释的。这些结果，再结合赖氨酸-67-谷氨酰胺突变体更严重的损伤，可以通过将赖氨酸-67侧链的正铵基团归因于静电作用来解释。
