20 research outputs found
Biochemical evidence for the tyrosine involvement in cationic intermediate stabilization in mouse β-carotene 15, 15'-monooxygenase
<p>Abstract</p> <p>Background</p> <p>β-carotene 15,15'-monooxygenase (BCMO1) catalyzes the crucial first step in vitamin A biosynthesis in animals. We wished to explore the possibility that a carbocation intermediate is formed during the cleavage reaction of BCMO1, as is seen for many isoprenoid biosynthesis enzymes, and to determine which residues in the substrate binding cleft are necessary for catalytic and substrate binding activity. To test this hypothesis, we replaced substrate cleft aromatic and acidic residues by site-directed mutagenesis. Enzymatic activity was measured <it>in vitro </it>using His-tag purified proteins and <it>in vivo </it>in a β-carotene-accumulating <it>E. coli </it>system.</p> <p>Results</p> <p>Our assays show that mutation of either Y235 or Y326 to leucine (no cation-π stabilization) significantly impairs the catalytic activity of the enzyme. Moreover, mutation of Y326 to glutamine (predicted to destabilize a putative carbocation) almost eliminates activity (9.3% of wt activity). However, replacement of these same tyrosines with phenylalanine or tryptophan does not significantly impair activity, indicating that aromaticity at these residues is crucial. Mutations of two other aromatic residues in the binding cleft of BCMO1, F51 and W454, to either another aromatic residue or to leucine do not influence the catalytic activity of the enzyme. Our <it>ab initio </it>model of BCMO1 with β-carotene mounted supports a mechanism involving cation-π stabilization by Y235 and Y326.</p> <p>Conclusions</p> <p>Our data are consistent with the formation of a substrate carbocation intermediate and cation-π stabilization of this intermediate by two aromatic residues in the substrate-binding cleft of BCMO1.</p
Evidence for environmentally coupled hydrogen tunneling during dihydrofolate reductase catalysis
Hydride transfer during catalysis by dihydrofolate reductase from Thermotoga maritima has been studied by stopped flow spectroscopy. The reduction of dihydrofolate by NADPH showed a biphasic temperature dependence of the deuterium kinetic isotope effect. At temperatures above 25 degrees C the KIE was temperature independent, while the reaction rates were strongly temperature dependent. Below 25 degrees C the KIE becomes dependent on temperature, and the ratio of the preexponential factors is inverse, suggesting a greater role for active dynamics that modulate the tunneling distance.status: publishe
6- and 14-Fluoro farnesyl diphosphate: Mechanistic probes for the reaction catalysed by aristolochene synthase
Differential specific radiation damage in the Cu-II-bound and Pd-II-bound forms of an alpha-helical foldamer: a case study of crystallographic phasing by RIP and SAD
The high photon flux at third-generation synchrotron sources can inflict significant primary radiation damage upon macromolecular crystals, even when the crystals are cryocooled. However, specific radiation-induced structural changes can be exploited for de novo phasing by an approach known as radiation damage-induced phasing (RIP). Here, RIP and single-wavelength anomalous dispersion (SAD) phasing were alternatively used to derive experimental phases to 1.2 Å resolution for crystals of an -helical 18-residue peptide, MINTS, which was derived from the neurotoxin apamin and the palladium-bound structure of which is now reported. Helix formation is induced by the binding of palladium (or copper) to two histidines spaced four residues apart, while two disulfide bonds tether the N-terminal helix to the C-terminal loop-like part of the peptide. Either RIP or SAD phasing of the palladium-bound and copper-bound forms of MINTS, which crystallized in different space groups, resulted in density maps of superb quality. Surprisingly, RIP phasing of the metal-bound complex structures of MINTS was a consequence of differential radiation damage, resting primarily on the reduction of the disulfide bonds in Pd-MINTS and on depletion of the metal sites in Cu-MINTS. Its miniprotein-like characteristics, versatile metal-binding properties and ease of crystallization suggest MINTS to be a convenient test specimen for methods development in crystallographic phasing based on either synchrotron or in-house X-ray diffraction data
Evidence that a ‘dynamic knockout’ in Escherichia coli dihydrofolate reductase does not affect the chemical step of catalysis
The question of whether protein motions play a role in the chemical step of enzymatic catalysis has generated much controversy in recent years. Debate has recently reignited over possible dynamic contributions to catalysis in dihydrofolate reductase, following conflicting conclusions from studies of the N23PP/S148A variant of the Escherichia coli enzyme. By investigating the temperature dependence of kinetic isotope effects, we present evidence that the reduction in the hydride transfer rate constants in this variant is not a direct result of impairment of conformational fluctuations. Instead, the conformational state of the enzyme immediately before hydride transfer, which determines the electrostatic environment of the active site, affects the rate constant for the reaction. Although protein motions are clearly important for binding and release of substrates and products, there appears to be no detectable dynamic coupling of protein motions to the hydride transfer step itself