M.P. Egloff

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTCutinase, a lipolytic enzyme with a preformed oxyanion holeChrislaine Martinez, Anne Nicolas, Herman van Tilbeurgh, Marie Pierre Egloff, Claire Cudrey, Robert Verger, and Christian CambillauCite this: Biochemistry 1994, 33, 1, 83–89Publication Date (Print):January 11, 1994Publication History Published online1 May 2002Published inissue 11 January 1994https://pubs.acs.org/doi/10.1021/bi00167a011https://doi.org/10.1021/bi00167a011research-articleACS PublicationsRequest reuse permissionsArticle Views745Altmetric-Citations148LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

Cutinases, a group of cutin degrading enzymes with molecular masses of around 22-25 kDa (Kolattukudy, 1984), are also able to efficiently hydrolyse triglycerides (De Geus et al., 1989; Lauwereys et al., 1991), but without exhibiting the interfacial activation phenomenom (Sarda et al., 1958). They belong to a class of proteins with a common structural framework, called the alpha/beta hydrolase fold (Martinez et al., 1992; Ollis et al., 1992). We describe herein the structure of cutinase covalently inhibited by diethyl-p-nitrophenyl phosphate (E600) and refined at 1.9-A resolution. Contrary to what has previously been reported with lipases (Brzozowski et al., 1991; Derewenda et al., 1992; Van Tilbeurgh et al., 1993), no significant structural rearrangement was observed here in cutinase upon the inhibitor binding. Moreover, the structure of the active site machinery, consisting of a catalytic triad (S120, H188, D175) and an oxyanion hole (Q121 and S42), was found to be identical to that of the native enzyme, whereas the oxyanion hole of Rhizomucor lipase (Brzozowski et al., 1991; Derewenda et al., 1992), like that of pancreatic lipase (van Tilbeurgh et al., 1993), is formed only upon enzyme-ligand complex formation. The fact that cutinase does not display interfacial activation cannot therefore only be due to the absence of a lid but might also be attributable to the presence of a preformed oxyanion hole.

Nucleoside analogues are currently used to treat human immunodeficiency virus infections. The appearance of up to five substitutions (A62V, V75I, F77L, F116Y, and Q151M) in the viral reverse transcriptase promotes resistance to these drugs, and reduces efficiency of the antiretroviral chemotherapy. Using pre-steady state kinetics, we show that Q151M and A62V/V75I/F77L/F116Y/Q151M substitutions confer to reverse transcriptase (RT) the ability to discriminate an analogue relative to its natural counterpart, and have no effect on repair of the analogue-terminated DNA primer. Discrimination results from a selective decrease of the catalytic rate constant k(pol): 18-fold (from 7 to 0.3 s(-1)), 13-fold (from 1.9 to 0.14 s(-1)), and 12-fold (from 13 to 1 s(-1)) in the case of ddATP, ddCTP, and 3'-azido-3'-deoxythymidine 5'-triphosphate (AZTTP), respectively. The binding affinities of the triphosphate analogues for RT remain unchanged. Molecular modeling explains drug resistance by a selective loss of electrostatic interactions between the analogue and RT. Resistance was overcome using alpha-boranophosphate nucleotide analogues. Using A62V/V75I/F77L/F116Y/Q151M RT, k(pol) increases up to 70- and 13-fold using alpha-boranophosphate-ddATP and alpha-boranophosphate AZTTP, respectively. These results highlight the general capacity of such analogues to circumvent multidrug resistance when RT-mediated nucleotide resistance originates from the selective decrease of the catalytic rate constant k(pol).