Pro167位点突变型CTX-M-14超广谱β-内酰胺酶的动力学特征

The characteristics of enzyme kinetics of CTX-M-14 type extended-spectrum β-lactamase with Pro167 residue substitution

目的 对Pr0167位点突变型CTX-M-14超广谱β-内酰胺酶（ESBL）的动力学特征进行分析与评价.方法 以携带CTX-M-14基因的大肠埃希菌临床株为模板,克隆目的 基因,重组工程菌,并表达CTX-M-14型ESBL.进而采用基于重叠延伸PCR法的定点突变技术,将CTX-M-14型ESBL的167位点Pro（P）分别突变为Gly（G）、Gln（Q）、Ser（S）和Thr（T）,重组构建P167G、P167Q、P167S和P167T四株突变型的CTX-M-14工程菌.表达与纯化野生型、重组型和突变型CTX-M-14型ESBL,检测其水解β-内酰胺类抗菌素的酶动力学参数（Kcat、Km和Km）.结果 对野生型与重组型CTX-M-14型ESBL的酶动力学参数进行配对t检验,结果显示两者Kcat（t=1.796,P=0.123）、Km（t=0.559,P=0.596）、Kcat/Km（t=0.893,P=0.406）间的差异无统计学意义（P〉0.1）.与重组CTX-M-14型ESBL相比,P167S突变型酶对头孢他啶的Km值大幅降低,为突变前的1/16（8.39/134.85）;Kcat和Km值分别为突变前的2.87倍（1.81/0.63）和43.6倍（0.218/0.005）;且对青霉素、氨苄西林、头孢唑啉、头孢呋辛、头孢曲松和头孢噻肟的Kcat/Km值都呈现出显著减小的趋势（P〈0.05）.与重组CTX-M-14 ESBL相比,P167Q和P167G突变型酶对头孢他啶的Kcat值亦大幅减小（P〈0.01）,而P167T突变型对头孢他啶的酶动力学参数无明显变化.结论 重组型与野生型CTX-M-14 ESBL之间各项酶动力学参数的差异无统计学意义（P〉0.1）.而P167S位点的突变,不仅增强了CTX-M-14型ESBL对头孢他啶的亲和力,也加快了酶-底物复合物的转换速率.通过对Pr0167位点4种突变型酶的动力学参数比较,初步说明突变型CTX-M ESBL对头孢他啶所具备的高水解活性机制,不能简单地归结于Pro167位点被小侧链氨基酸残基取代而导致酶活性中心空间扩大的解释.

Objective To analyze and evaluate the characteristics of enzyme kinetics of CTX-M-14 type extended-spectrum β-lactamase（ESBL） with Pro167 residue substitution. Methods By molecular cloning and PCR techniques, CTX-M-14 gene was directionally cloned into plasmid pET28a（ ＋ ） from a clinical E. coli isolate and formed an expression vector to transform competent E. coli BL21 （DE3 ）. Prol67 residue substitutions of P167G, P167Q, P167S and P167T were introduced to CTX-M-14 by site-directed muta-genesis based on overlap extension PCR with the former recombinant plasmid as PCR template, respectively.The wild-type CTX-M-14, recombinant CTX-M-14 protein and its variants were expressed and purified, then their steady-state kinetic parameters （Kcat, Km and Kcat/Km ） against β-lactam antibiotics were determined.Results The kinetic parameters of wild-type and recombinant CTX-M-14 had no statistically significant differences （P〉0.1）. The 1/Km, Kcat and Kcat/Km values of P167S variant against ceftazidime were 16-fold, 2.87-fold and 43.6-fold higher than those of recombinant CTX-M-14, respectively, and the Kcat/Km value of P167S variant against penicillin, ampicillin, cefazolin, cefuroxime, ceftriaxone, cefotaxime decreased（ 〈 0.05）. Compared with the kinetic parameters of recombinant CTX-M-14, the kinetic parameters of P167T variant against ceftazidime had no significant change, but the Kcat values of P167Q and P167G variants decreased dramatically（P〈0.01）. Conclusion There was no difference between the enzyme activities of wild-type and recombinant CTX-M-14. P167S variant could not only promote the enzyme affinity of CTX-M-14 to ceftazidime but also improve the conversion rate of enzyme-substrate complex in the ceftazidime hydrolysis. The comparison of the kinetic parameters of CTX-M-14 and its variants with Pro167 residue substitution showed that the increased activity of CTX-M ESBL variants against ceftazidime could not be simply explained with the enlarged cavity in active site that may be caused by the replacement of Pro167 residue by smaller amino acids.