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John P Evans

Publications and source records attributed to John P Evans.

8 recordsLinked to original sources
PubMed2006

The catalytic role of the copper ligand H172 of peptidylglycine alpha-hydroxylating monooxygenase: a kinetic study of the H172A mutant.

An essential histidine ligand to the electron transfer copper (CuH) of peptidylglycine alpha-hydroxylating monooxygenase (PHMcc) was mutated to an alanine and found to retain copper binding and hydroxylase activity [Jaron, S., et al. (2002) Biochemistry 41, 13274-13282]. An extensive kinetic and deuterium isotope effect study finds this mutant to maintain full coupling of O2 consumed to product formed despite a 3 order-of-magnitude decrease in kcat and a 300-fold decrease in kcat/Km(O2). Unexpectedly, electron transfer is not rate-limiting in H172A. Rather, the increased kinetic isotope effect (KIE) on kcat of 3.27 +/- 0.39 suggests that C-H bond cleavage has become more rate-limiting, implicating a role for His172 that goes beyond that of a simple ligand to CuH. The mechanistic implications are discussed.

Alanine↗
PubMed2006

Motexafin gadolinium-induced cell death correlates with heme oxygenase-1 expression and inhibition of P450 reductase-dependent activities.

Heme oxygenase-1 (HO1), which oxidizes heme to biliverdin, CO, and free iron, conveys protection against oxidative stress and is antiapoptotic. Under stress conditions, some porphyrin derivatives can inhibit HO1 and trigger cell death. Motexafin gadolinium (MGd) is an expanded porphyrin that selectively targets cancer cells through a process of futile redox cycling that decreases intracellular reducing metabolites and protein thiols. Here, we report that hematopoietic-derived cell lines that constitutively express HO1 are more susceptible to MGd-induced apoptosis than those that do not. MGd used in combination with tin protoporphyrin IX, an inhibitor of HO1, resulted in synergistic cell killing. Consistent with these cell culture observations, we found that MGd is an inhibitor of heme oxygenase-1 activity in vitro. We demonstrate that inhibition of HO1 reflects an interaction of MGd with NADPH-cytochrome P450 reductase, the electron donor for HO1, that results in diversion of reducing equivalents from heme oxidation to oxygen reduction. In accord with this mechanism, MGd is also an in vitro inhibitor of CYP2C9, CYP3A4, and CYP4A1. Inhibition of HO1 by MGd may contribute to its anticancer activity, whereas its in vitro inhibition of a broad spectrum of P450 enzymes indicates that a potential exists for drug-drug interactions.

Antineoplastic Agents↗
PubMed2006

Alteration of the regiospecificity of human heme oxygenase-1 by unseating of the heme but not disruption of the distal hydrogen bonding network.

Heme oxygenase regiospecifically oxidizes heme at the alpha-meso position to give biliverdin IXalpha, CO, and iron. The heme orientation within the active site, which is thought to determine the oxidation regiospecificity, is shown here for the human enzyme (hHO1) to be largely determined by interactions between the heme carboxylic acid groups and residues Arg183 and Lys18 but not Tyr134. Mutation of either Arg183 or Lys18 individually does not significantly alter the NADPH-cytochrome P450 reductase-dependent reaction regiochemistry but partially shifts the oxidation to the beta/delta-meso positions in the reaction supported by ascorbic acid. Mutation of Glu29 to a lysine, which places a positive charge where it can interact with a heme carboxyl if the heme rotates by approximately 90 degrees, causes a slight loss of regiospecificity but combined with the R183E and K18E mutations results primarily in beta/delta-meso oxidation of the heme under all conditions. NMR analysis of heme binding to the triple K18E/E29K/R183E mutant confirms rotation of the heme in the active site. Kinetic studies demonstrate that mutations of Arg183 greatly impair the rate of the P450 reductase-dependent reaction, in accord with the earlier finding that Arg183 is involved in binding of the reductase to hHO1, but have little effect on the ascorbate reaction. Mutations of Asp140 and Tyr58 that disrupt the active site hydrogen bonding network impair catalytic rates but do not influence the oxidation regiochemistry. The results indicate both that the oxidation regiochemistry is largely controlled by ionic interactions of the heme propionic acid groups with the protein and that shifts in regiospecificity involve rotation of the heme about an axis perpendicular to the heme plane.

Arginine↗
PubMed2004

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Conflict of Interest↗
PubMed2003

Evidence that dioxygen and substrate activation are tightly coupled in dopamine beta-monooxygenase. Implications for the reactive oxygen species.

Oxygen activation occurs at a wide variety of enzyme active sites. Mechanisms previously proposed for the copper monooxygenase, dopamine beta-monooxygenase (DbetaM), involve the accumulation of an activated oxygen intermediate with the properties of a copper-peroxo or copper-oxo species before substrate activation. These are reminiscent of the mechanism of cytochrome P-450, where a heme iron stabilizes the activated O2 species. Herein, we report two experimental probes of the activated oxygen species in DbetaM. First, we have synthesized the substrate analog, beta,beta-difluorophenethylamine, and examined its capacity to induce reoxidation of the prereduced copper sites of DbetaM upon mixing with O2 under rapid freeze-quench conditions. This experiment fails to give rise to an EPR-detectable copper species, in contrast to a substrate with a C-H active bond. This indicates either that the reoxidation of the enzyme-bound copper sites in the presence of O2 is tightly linked to C-H activation or that a diamagnetic species Cu(II)-O2* has been formed. In the context of the open and fully solvent-accessible active site for the homologous peptidylglycine-alpha-hydroxylating monooxygenase and by analogy to cytochrome P-450, the accumulation of a reduced and activated oxygen species in DbetaM before C-H cleavage would be expected to give some uncoupling of oxygen and substrate consumption. We have, therefore, examined the degree to which O2 and substrate consumption are coupled in DbetaM using both end point and initial rate experimental protocols. With substrates that differ by more than three orders of magnitude in rate, we fail to detect any uncoupling of O2 uptake from product formation. We conclude that there is no accumulation of an activated form of O2 before C-H abstraction in the DbetaM and peptidylglycine-alpha-hydroxylating monooxygenase class of copper monooxygenases, presenting a mechanism in which a diamagnetic Cu(II)-superoxo complex, formed initially at very low levels, abstracts a hydrogen atom from substrate to generate Cu(II)-hydroperoxo and substrate-free radical as intermediates. Subsequent participation of the second copper site per subunit completes the reaction cycle, generating hydroxylated product and water.

Adrenal Glands↗