Biochemical Characterization and Structural Analysis of a Highly Proficient Cocaine Esterase

详细信息    查看全文

• 作者：James M. Turner ; Nicholas A. Larsen ; Amrik Basran ; Carlos F. Barbas ; III ; Neil C. Bruce ; Ian A. Wilson ; and Richard A. Lerner
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：October 15, 2002
• 年：2002
• 卷：41
• 期：41
• 页码：12297 - 12307
• 全文大小：600K
• 年卷期：v.41,no.41(October 15, 2002)
• ISSN：1520-4995

文摘

The bacterial cocaine esterase, cocE, hydrolyzes cocaine faster than any other reported cocaineesterase. Hydrolysis of the cocaine benzoyl ester follows Michaelis-Menten kinetics with k_cat = 7.8 s^-1and K_M = 640 nM. A similar rate is observed for hydrolysis of cocaethylene, a more potent cocainemetabolite that has been observed in patients who concurrently abuse cocaine and alcohol. The highcatalytic proficiency, lack of observable product inhibition, and ability to hydrolyze both cocaine andcocaethylene make cocE an attractive candidate for rapid cocaine detoxification in an emergency setting.Recently, we determined the crystal structure of this enzyme, and showed that it is a serine carboxylesterase,with a catalytic triad formed by S117, H287, and D259 within a hydrophobic active site, and an oxyanionhole formed by the backbone amide of Y118 and the Y44 hydroxyl. The only enzyme previously knownto use a Tyr side chain to form the oxyanion hole is prolyl oligopeptidase, but the Y44F mutation of cocEhas a more deleterious effect on the specificity rate constant (k_cat/K_M) than the analogous Y473F mutationof prolyl oligopeptidase. Kinetic studies on a series of cocE mutants both validate the proposed mechanism,and reveal the relative contributions of active site residues toward substrate recognition and catalysis.Inspired by the anionic binding pocket of the cocaine binding antibody GNC92H2, we found that a Q55Emutation within the active site of cocE results in a modest (2-fold) improvement in K_M, but a 14-fold lossof k_cat. The pH rate profile of cocE was fit to the ionization of two groups (pK_a1 = 7.7; pK_a2 = 10.4) thatlikely represent titration of H287 and Y44, respectively. We also describe the crystal structures of bothS117A and Y44F mutants of cocE. Finally, urea denaturation studies of cocE by fluorescence and circulardichroism show two unfolding transitions (0.5-0.6 M and 3.2-3.7 M urea), with the first transitionlikely representing pertubation of the active site.