Enzymatic Methyl Transfer: Role of an Active Site Residue in Generating Active Site Compaction That Correlates with Catalytic Efficiency

Human catechol-<i>O</i>-methyltransferase (COMT) catalyzes a methyl transfer from <i>S</i>-adenosylmethionine (AdoMet) to dopamine. Site-specific mutants at three positions (Tyr68, Trp38, and Val108) have been characterized with regard to product distribution, catalytic efficiency, and secondary kinetic isotope effects. The series of mutations at Tyr68 within wild-type protein and the common polymorphic variant (Val108Met) yields a linear correlation between the catalytic efficiency and the size of the secondary kinetic isotope effect. We conclude that active site compaction in COMT is modulated by a proximal side chain residing behind the sulfur-bearing methyl group of AdoMet. These findings are discussed in the context of the active site compression that has been postulated to accompany enzyme-supported hydrogen tunneling

Kinetic Detection of Orthogonal Protein and Chemical Coordinates in Enzyme Catalysis: Double Mutants of Soybean Lipoxygenase

Soybean lipoxygenase-1 (SLO-1) is a paradigmatic enzyme system for studying the contribution of hydrogen tunneling to enzymatic proton-coupled electron transfer processes. In this study, the impact of pairs of double mutants on the properties of SLO-1 is presented. Steady-state rates and their deuterium kinetic isotope effects (KIEs) have been measured for the bimolecular reaction of enzyme with free substrate (<i>k</i>_cat/<i>K</i>_m) and compared to the unimolecular rate constant, <i>k</i>_cat. A key kinetic finding is that the competitive KIEs on the second-order rate constant (<i>k</i>_cat/<i>K</i>_m) are all reduced from ^D<i>k</i>_cat and, despite large changes in rate and activation parameters, remain essentially unaltered under a variety of conditions. These data implicate a protein reaction coordinate that is <i>orthogonal</i> to the chemical reaction coordinate and controls the concentration of the active enzyme. This study introduces a new means to interrogate the alteration of conformational landscapes that can occur following site-specific mutagenesis

Hydrogen Tunneling in a Prokaryotic Lipoxygenase

A bacterial lipoxygenase (LOX) shows a deuterium kinetic isotope effect (KIE) that is similar in magnitude and temperature dependence to the very large KIE of eukaryotic LOXs. This occurs despite the evolutionary distance, an ∼25% smaller catalytic domain, and an increase in <i>E</i>_a of ∼11 kcal/mol. Site-specific mutagenesis leads to a protein variant with an <i>E</i>_a similar to that of the prototypic plant LOX, providing possible insight into the origin of evolutionary differences. These findings, which extend the phenomenon of hydrogen tunneling to a prokaryotic LOX, are discussed in the context of a role for protein size and/or flexibility in enzymatic hydrogen tunneling

Purification of human AOC3 expressed by s2 <i>Drosophila</i> cells.

<p>A. Immunoblot of fractions obtained after ion exchange column chromatography, showing human AOC3 at the expected mass. Protein ladder to the left of Lane 1 (top band, blue −100 kDa; bottom band, red −75 kDa). B. 10% acrylamide, denaturing SDS-PAGE gel electrophoresis of fractions using a Laemmli buffering system after gel filtration column chromatography. Fractions represented in lanes 4 to 8 and 10 were isolated and concentrated for further characterization. Protein ladder shown in Lane 9 (second band from top −116 kDa, third band −97 kDa, fourth band −66 kDa). Approximately 0.25–2 ug of protein was loaded into each lane.</p

Comparison of second order rate constants (<i>k</i>_cat/K_m) between purified human and mouse AOC3.

<p>^<i>a</i><i>k</i>_cat/K_m (M⁻¹ s⁻¹)×10³ for human and mouse AOC3.</p

Comparison of AOC3 K_m values determined by steady state kinetic studies of purified murine enzyme and whole cell 3T3-L1 adipocytes.

<p>^<i>a</i>K_m value calculated by plotting endpoint rates vs. substrate concentration, correcting for baseline Amplex Red oxidation by adipocytes pre-treated with 1 mM semicarbazide for 30 min.</p><p>^<i>b</i>K_m values are averages derived from both endpoint fluorescence versus substrate concentration and linear rates of Amplex Red oxidation versus substrate concentration.</p

Human AOC3 substrate kinetic profile.

<p>^<i>a</i>Substrates of human AOC3 with entries found in the Human Metabolome Database version 2.5.</p><p>^<i>b</i>Substrate kinetics were measured at approximately 19 to 23% air (4 to 5% O₂).</p

Condensation of acetyl-CoA and glycine to form the intermediate, 2-amino 3-ketobutyrate, a precursor of aminoacetone formation.

<p>Threonine dehydrogenase (TDH) normally catalyzes the reduction of 2-amino 3-ketobutyrate to form threonine and prevents its buildup; however, TDH is an inactive pseudo-gene in humans.</p

Adipocyte cell culture-based determination of AOC3 turnover in the presence of varying concentrations of methylamine substrate using the Amplex Red peroxide detection assay.

<p>Adipocyte cell culture-based determination of AOC3 turnover in the presence of varying concentrations of methylamine substrate using the Amplex Red peroxide detection assay.</p

Properties of purified human AOC3.

a<p>Per monomer.</p>b<p>At ambient O₂, pH 7.4, 37°C.</p