Investigating the catalytic role of Cys150‐Asp29‐Lys117 in maleamate amidohydrolase

Abstract

Maleamate amidohydrolase (NicF) catalyzes the penultimate reaction in the bacterial oxidation of nicotinate to fumarate. On the basis of sequence homology and structural data, catalysis of maleamate hydrolysis to maleate and ammonia by NicF has been proposed to proceed by a nucleophilic addition‐elimination mechanism involving the thiolate side‐chain of C150. In support of that proposal, NicF activity is shown to be inactivated by iodoacetamide and reversibly inactivated by MMTS. Kinetic analyses of C150S and C150A variants of NicF show a 105–106‐fold drop in kcat, without affecting KM, providing additional functional evidence in support of a critical role in catalysis by C150. Likewise, mutations of D29 or K117, the other two conserved residues that complete a proposed catalytic triad, also produce NicF variants with significant drops in kcat (103–104‐fold). Effects of changing pH (4.0–11.0) on kcat and kcat/KM of wtNicF, and the pH dependence of the rate of wt NicF inhibition by iodoacetamide, both suggest the involvement of two general A/B catalysts. Inhibition of wt NicF by 5‐hydroxyfuran‐2(5H)‐one is competitive and reversible with a Ki of 139 nM, consistent with the formation of a quasi‐stable thiohemiacetal that mimics the structure of the proposed intermediates along the reaction coordinate. This work was supported by a HHMI Undergraduate Science Education Program Award to the College of Wooster.