Insights on the Mechanism of Amine Oxidation Catalyzed by d-Arginine Dehydrogenase Through pH and Kinetic Isotope Effects

Abstract

The mechanism of amine oxidation catalyzed by D-arginine dehydrogenase (DADH) has been investigated using steady-state and rapid reaction kinetics, with pH, substrate and solvent deuterium kinetic isotope effects (KIE) as mechanistic probes, and computational studies. Previous results showed that 85-90% of the flavin reduction reaction occurs in the mixing time of the stopped-flow spectrophotometer when arginine is the substrate, precluding a mechanistic investigation. Consequently, leucine, with slower kinetics, has been used here as the flavin-reducing substrate. Free energy calculations and the pH profile of the K(d) are consistent with the enzyme preferentially binding the zwitterionic form of the substrate. Isomerization of the Michaelis complex, yielding an enzyme-substrate complex competent for flavin reduction, is established due to an inverse hyperbolic dependence of k(cat)/K(m) on solvent viscosity. Amine deprotonation triggers the oxidation reaction, with cleavage of the substrate NH and CH bonds occurring in an asynchronous fashion, as suggested by the multiple deuterium KIE on the rate constant for flavin reduction (k(red)). A pK(a) of 9.6 signifies the ionization of a group that facilitates flavin reduction in the unprotonated form. The previously reported high-resolution crystal structures of the iminoarginine and iminohistidine complexes of DADH allow us to propose that Tyr(53), on a mobile loop covering the active site, may participate in substrate binding and facilitate flavin reduction.

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

“…Free energy calculations and the pH profile for K d are consistent with the enzyme binding preferentially the zwitterionic form of Dleucine [7]. After isomerization of the enzyme-substrate complex, amine deprotonation triggers the oxidation reaction, with cleavages of the NH and CH bonds of D-leucine occurring asynchronously, as suggested by multiple deuterium kinetic isotope effects on the rate constant for flavin reduction [7]. In this respect PaDADH is similar to proline dehydrogenase [8], but different from other flavin-dependent amine oxidases, such as DAAO or monomeric sarcosine oxidase, for which amine oxidation occurs from the anionic substrate [9].…”

“…This conclusion comes from the pH-independence of the D k red value for the reduction of the enzyme-bound flavin with D-leucine between pH 7.0 and 10.3, where both k cat and k cat /K m depend on pH, which establishes that the enzyme-substrate complex is not committed to catalysis and that D-leucine is not a sticky substrate [24]. Independent support for D-leucine being a slow substrate is provided by the comparison of the pK a of $9.5 determined for k cat /K m with the pK a of 9.6 recently reported for the k red pH-profile with D-leucine [7]. The active site of the enzyme has several side chains that could potentially act as catalytic base for the deprotonation of the a-NH 3 + group of the amino acid substrate, including H48, Y53, Y249 and, although less likely due to its location and participation in binding the side chain of cationic substrate, E87.…”

“…The concentration of PMS was kept fixed at 1 mM to ensure full saturation of the enzyme. Data for D-leucine with the wild-type were taken from Yuan et al [7].…”

“…Amine oxidation has been http://dx.doi.org/10.1016/j.abb.2015.01.017 0003-9861/Ó 2015 Elsevier Inc. All rights reserved. studied with D-leucine, which is amenable to mechanistic investigation using a stopped-flow spectrophotometer, with pH, substrate and solvent kinetic isotope effects, as well as computational studies [7]. Free energy calculations and the pH profile for K d are consistent with the enzyme binding preferentially the zwitterionic form of Dleucine [7].…”

“…studied with D-leucine, which is amenable to mechanistic investigation using a stopped-flow spectrophotometer, with pH, substrate and solvent kinetic isotope effects, as well as computational studies [7]. Free energy calculations and the pH profile for K d are consistent with the enzyme binding preferentially the zwitterionic form of Dleucine [7]. After isomerization of the enzyme-substrate complex, amine deprotonation triggers the oxidation reaction, with cleavages of the NH and CH bonds of D-leucine occurring asynchronously, as suggested by multiple deuterium kinetic isotope effects on the rate constant for flavin reduction [7].…”