Ball Jacob, Bui Quan V V, Gannavaram Swathi, Gadda Giovanni
Department of Chemistry, Georgia State University, Atlanta, GA 30302-3965, United States.
Department of Chemistry, Georgia State University, Atlanta, GA 30302-3965, United States; Georgia Perimeter College, Decatur, GA 30034-3832, United States.
Arch Biochem Biophys. 2015 Feb 15;568:56-63. doi: 10.1016/j.abb.2015.01.017. Epub 2015 Jan 28.
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 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 (D)kred 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.
铜绿假单胞菌D-精氨酸脱氢酶（PaDADH）催化D-精氨酸氧化为亚氨基精氨酸，亚氨基精氨酸会非酶水解为2-酮精氨酸和氨。在此，通过定点诱变和pH效应来研究该酶对两性离子和阳离子底物的结合及催化作用。结合D-精氨酸或D-赖氨酸需要一个表观pKa值⩾7.9的未质子化基团，但结合D-甲硫氨酸或D-亮氨酸则不需要。这个基团是E87，用亮氨酸取代它可证明这一点。所有底物进行胺氧化都需要一个pKa为9.5的未质子化基团，该基团在H48F和E87L变体中仍然存在。由于之前已排除Y53和Y249，所以将该pKa值归因于底物α-NH3(+)基团，之前的量子力学/分子力学（QM/MM）和解离常数（Kd）pH曲线表明该基团质子化有利于与酶结合。对D-亮氨酸的（D）kred没有pH效应，确定9.5为固有pKa值，且D-亮氨酸为非粘性底物。D-精氨酸、D-赖氨酸和D-甲硫氨酸及其相应的亚氨基产物比D-亮氨酸粘性大得多，kcat/Km和kcat中的表观pKa值<9.5表明了这一点。通过野生型PaDADH与D-赖氨酸以及H48F和E87L酶与D-精氨酸的空心kcat pH曲线确定了催化过程中质子移动受限。
