Mutation of a distal gating residue modulates NADH binding in NADH:Quinone oxidoreductase from Pseudomonas aeruginosa PAO1

Targeted Mutation of a Non-catalytic Gating Residue Increases the Rate of Pseudomonas aeruginosa d-Arginine Dehydrogenase Catalytic Turnover

“…Upon E 246 replacement with glutamine, glycine, or leucine, the hydrogen bond interactions with the Y 53 gate of loop L1 are hindered, likely leading to altered loop motions as recently reported for the P. aeruginosa NADH:quinone oxidoreductase and pyranose 2-oxidase when the Q 80 and F 454 gating loop residues were respectively mutated. , …”

“…The decreased rate of hydride transfer might be due to a different configuration or a decreased probability of the enzyme–substrate (ES) complex competent for the hydride transfer reaction upon mutation of the E 246 residue. The increased K d values are expected for mutating a loop residue that controls the active site gate because the removal of the residue renders the active site more open, leading to increased rates of substrate dissociation. The increased rate of substrate dissociation is picked up by the 2–4-fold lower second-order rate constant k cat / K m , which measures the ability of the enzyme to bind the free substrate to form the enzyme–substrate complex that undergoes catalysis, as observed for both the d -leucine and d -arginine substrates (Tables and ).…”

“…Upon E 246 replacement with glutamine, glycine, or leucine, the hydrogen bond interactions with the Y 53 gate of loop L1 are hindered, likely leading to altered loop motions as recently reported for the P. aeruginosa NADH:quinone oxidoreductase and pyranose 2-oxidase when the Q 80 and F 454 gating loop residues were respectively mutated. 20,50 For the E 246 G variant enzyme with D-leucine as a substrate, using eq 3, a rate constant for the iminoleucine release k P-rel of ∼188 ± 7 s −1 could be estimated from the k red and k cat values of the enzyme (Table 2). Such a rate is only ∼4× faster than the rate of catalysis for the enzyme, consistent with both hydride transfer and product release contributing toward the overall turnover of the enzyme.…”

“…Loops, the most flexible secondary structural elements of enzymes, play significant roles in enzyme catalysis. Loops mediate substrate binding, maintain the integrity of the active site, protect from the bulk solvent after enzyme–substrate complex formation, and initiate conformational changes. − Multiple studies demonstrate how loop modulations can alter the catalytic function of an enzyme. ,− In a site-directed mutagenesis study that investigates the roles of loop residues A 85 and I 86 located at the binding pocket of Thermoanaerobacter brockii alcohol dehydrogenase, the engineered enzyme variants A 85 G and I 86 L gained the ability to reduce bulky ketones to the corresponding alcohols with high enantioselectivities . Similarly, a study on cumene dioxygenase demonstrates how site-directed mutageneses of multiple critical active site loop residues resulted in significant improvement of product formation with alterations in the regioselectivity and enantioselectivity of the enzyme .…”

“…Enzymes are highly efficient macromolecules that catalyze many biochemical reactions in living cells. − Enzymes assume multiple conformational changes during catalysis and can accelerate reactions by many orders of magnitude. − During enzyme engineering, scientists often aim to increase the overall rate of enzyme turnover, k cat , which, unless demonstrated otherwise, might be limited by the chemical step of catalysis and the mechanical step of product release enhanced by favorable enzyme motions. − However, there is a tendency for engineers to consider the k cat parameter as a single kinetic step, which may limit the approaches used in protein engineering. Scientists do not fully understand the effects of enzyme motions on enzyme catalysis, such as loop dynamics.…”