Importance of glutamate 87 and the substrate α-amine for the reaction catalyzed by d-arginine dehydrogenase

•Restricted proton movements are established in catalysis from hollowed pH profiles.•E87 (pKa ⩾ 7.9) is required to bind cationic but not zwitterionic substrates.•The unprotonated substrate α-amine (pKa ∼ 9.5) is required for substrate oxidation.•Leucine is a non-sticky substrate for the enzyme.•Arginine, lysine and methionine are sticky substrates for the enzyme.

Pseudomonas aeruginosad-arginine dehydrogenase (PaDADH) catalyzes the oxidation of d-arginine to iminoarginine, which is non-enzymatically hydrolyzed to 2-ketoarginine and ammonia. Here, site-directed mutagenesis and pH effects were used to investigate binding and catalysis of zwitterionic and cationic substrates for the enzyme. An unprotonated group with apparent pKa value ⩾7.9 is required for binding d-arginine or d-lysine, but not d-methionine or d-leucine. This group is E87, as suggested by its replacement with leucine. An unprotonated group with pKa of 9.5, which persists in the H48F and E87L variants, is required for amine oxidation with all substrates. Since Y53 and Y249 were previously ruled out, the pKa is assigned to the substrate α-NH3+ group, which previous QM/MM and Kd pH-profile demonstrated to be protonated for preferred binding to the enzyme. Lack of pH effects on the Dkred with d-leucine established 9.5 as the intrinsic pKa, and d-leucine as a non-sticky substrate. d-Arginine, d-lysine and d-methionine and their corresponding iminoproducts were significantly stickier than d-leucine, as indicated by apparent pKa values <9.5 in both kcat/Km and kcat. Restricted proton movements in catalysis were established from hollowed kcat pH profiles in wild-type PaDADH with d-lysine and in the H48F and E87L enzymes with d-arginine.

Graphical abstractFigure optionsDownload full-size imageDownload high-quality image (98 K)Download as PowerPoint slide