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

Abstract Pseudomonas aeruginosa d -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 p K a 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 p K a 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 p K a is assigned to the substrate α-NH 3 + group, which previous QM/MM and K d pH-profile demonstrated to be protonated for preferred binding to the enzyme. Lack of pH effects on the D k red with d -leucine established 9.5 as the intrinsic p K a , 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 p K a values k cat / K m and k cat . Restricted proton movements in catalysis were established from hollowed k cat pH profiles in wild-type PaDADH with d -lysine and in the H48F and E87L enzymes with d -arginine.