Wang Xiaojun, Minasov George, Shoichet Brian K
Department of Molecular Pharmacology and Biological Chemistry, Northwestern University School of Medicine, 303 East Chicago Avenue, Chicago, IL 60611-3008, USA.
J Mol Biol. 2002 Jun 28;320(1):85-95. doi: 10.1016/S0022-2836(02)00400-X.
Pressured by antibiotic use, resistance enzymes have been evolving new activities. Does such evolution have a cost? To investigate this question at the molecular level, clinically isolated mutants of the beta-lactamase TEM-1 were studied. When purified, mutant enzymes had increased activity against cephalosporin antibiotics but lost both thermodynamic stability and kinetic activity against their ancestral targets, penicillins. The X-ray crystallographic structures of three mutant enzymes were determined. These structures suggest that activity gain and stability loss is related to an enlarged active site cavity in the mutant enzymes. In several clinically isolated mutant enzymes, a secondary substitution is observed far from the active site (Met182-->Thr). This substitution had little effect on enzyme activity but restored stability lost by substitutions near the active site. This regained stability conferred an advantage in vivo. This pattern of stability loss and restoration may be common in the evolution of new enzyme activity.
在抗生素使用的压力下，耐药酶不断进化出新的活性。这种进化有代价吗？为了在分子水平上研究这个问题，对临床分离的β-内酰胺酶TEM-1突变体进行了研究。纯化后的突变酶对头孢菌素抗生素的活性增加，但对其祖先靶点青霉素失去了热力学稳定性和动力学活性。测定了三种突变酶的X射线晶体结构。这些结构表明，活性增加和稳定性丧失与突变酶中扩大的活性位点腔有关。在几种临床分离的突变酶中，在远离活性位点的位置观察到一个二级取代（Met182→Thr）。这种取代对酶活性影响很小，但恢复了因活性位点附近的取代而丧失的稳定性。这种恢复的稳定性在体内赋予了一种优势。这种稳定性丧失和恢复的模式可能在新酶活性的进化中很常见。
