Mechanistic analysis of NADH peroxidase

Abstract

NADH peroxidase (EC 1.11.1.1.) isolated from Strepococcus faecalis 1OC1 is an FAD-dependent protein with catalyzes the reation: NADH + H{dollar}\sp+{dollar} + H{dollar}\sb2{dollar}O{dollar}\sb2{dollar} {dollar}\to{dollar} NAD{dollar}\sp+{dollar} 2H{dollar}\sb2{dollar}O.;The steady state kinetic mechanism has been determined to be ping-pong. Pyridine nucleotide oxidation occurs via transfer of the 4S hydrogen atom to FAD. The specificity of the enzyme for 12 alternate nucleotide substrates has been determined, and a reductive half-reaction scheme has been proposed in which NADH is bound to both the oxidized and two-electron reduced forms of NADH peroxidase.;Primary deuterium and D{dollar}\sb2{dollar}O solvent kinetic isotope effects have been determined. The pH profiles and the pH dependence of the primary deuterium kinetic isotope effects have also been determined. These data indicated that there was an enzymic group with a pK = 7.2 that, when protonated, increases both the rate of reaction and the values of the primary dueterium kinetic isotope effect on V ({dollar}\sp{lcub}\rm D{rcub}{dollar}V) exhibited by NADH. These findings in conjunction with the D{dollar}\sb2{dollar}O solvent data suggest that there is one proton involved in the rate limiting-step in the oxidative half reaction. A chemical mechanism has been proposed for peroxide cleavage based on these data.;The enzyme has been purified to homogeneity for structural analysis. Extensive attempts to alkylate the active site cysteine(s) with {dollar}\sp3{dollar}H-iodoacetic acid were performed, however these attempts were unsuccessful. Purified enzyme was digested with trypsin, and several resultant peptides were sequenced. The first 40 amino acids of the amino-terminus have been sequenced.;Crystals of NADH peroxidase have been grown by the hanging drop vapor diffusion method in (NH{dollar}\sb4{dollar}){dollar}\sb2{dollar}SO{dollar}\sb4{dollar}. The unit cell axes are a = 76.6A, b = 132.9A, and c = 145.7A. The crystals diffract in the X-ray beam for several days, are catalytically active, and diffract to 2.5A resolution. Recently a search for heavy atom derivatives has begun. Crystals equilibrated with solutions containing C{dollar}\sb2{dollar}H{dollar}\sb5{dollar}HgPO{dollar}\sb4{dollar}, Pt(H{dollar}\sb2{dollar}NCH{dollar}\sb2{dollar}CH{dollar}\sb2{dollar}NH{dollar}\sb2{dollar})Cl{dollar}\sb2{dollar}, AuCl{dollar}\sb4{dollar}, and K{dollar}\sb3{dollar}UO{dollar}\sb2{dollar}F{dollar}\sb5{dollar} have been examined.;Attempt to deduce the amino acid sequence of the protein have begun. Early experiments to screen a genomic library with 24 and 42 base degenerate oligonucleotides derived from the amino-terminal amino acid sequence were inconclusive. However, progress has been made with two oligonucleotides designed for use in the polymerase chain reaction. Using this technique several portions of S. faecalis DNA have been amplified.