Biomedical subjects

Lawrence J Hayward

Publications and source records attributed to Lawrence J Hayward.

4 recordsLinked to original sources

PubMed2003

An alternative mechanism of bicarbonate-mediated peroxidation by copper-zinc superoxide dismutase: rates enhanced via proposed enzyme-associated peroxycarbonate intermediate.

Hydrogen peroxide can interact with the active site of copper-zinc superoxide dismutase (SOD1) to generate a powerful oxidant. This oxidant can either damage amino acid residues at the active site, inactivating the enzyme (the self-oxidative pathway), or oxidize substrates exogenous to the active site, preventing inactivation (the external oxidative pathway). It is well established that the presence of bicarbonate anion dramatically enhances the rate of oxidation of exogenous substrates. Here, we show that bicarbonate also substantially enhances the rate of self-inactivation of human wild type SOD1. Together, these observations suggest that the strong oxidant formed by hydrogen peroxide and SOD1 in the presence of bicarbonate arises from a pathway mechanistically distinct from that producing the oxidant in its absence. Self-inactivation rates are further enhanced in a mutant SOD1 protein (L38V) linked to the fatal neurodegenerative disorder, familial amyotrophic lateral sclerosis. The 1.4 A resolution crystal structure of pathogenic SOD1 mutant D125H reveals the mode of oxyanion binding in the active site channel and implies that phosphate anion attenuates the bicarbonate effect by competing for binding to this site. The orientation of the enzyme-associated oxyanion suggests that both the self-oxidative and external oxidative pathways can proceed through an enzyme-associated peroxycarbonate intermediate.

Amyotrophic Lateral Sclerosis↗

PubMed2002

Familial amyotrophic lateral sclerosis mutants of copper/zinc superoxide dismutase are susceptible to disulfide reduction.

We observed that 14 biologically metallated mutants of copper/zinc superoxide dismutase (SOD1) associated with familial amyotrophic lateral sclerosis all exhibited aberrantly accelerated mobility during partially denaturing PAGE and increased sensitivity to proteolytic digestion compared with wild type SOD1. Decreased metal binding site occupancy and exposure to the disulfide-reducing agents dithiothreitol, Tris(2-carboxyethyl)phosphine (TCEP), or reduced glutathione increased the fraction of anomalously migrating mutant SOD1 proteins. Furthermore, the incubation of mutant SOD1s with TCEP increased the accessibility to iodoacetamide of cysteine residues that normally participate in the formation of the intrasubunit disulfide bond (Cys-57 to Cys-146) or are buried within the core of the beta-barrel (Cys-6). SOD1 enzymes in spinal cord lysates from G85R and G93A mutant but not wild type SOD1 transgenic mice also exhibited abnormal vulnerability to TCEP, which exposed normally inaccessible cysteine residues to modification by maleimide conjugated to polyethylene glycol. These results implicate SOD1 destabilization under cellular disulfide-reducing conditions at physiological pH and temperature as a shared property that may be relevant to amyotrophic lateral sclerosis mutant neurotoxicity.

Amino Acid Substitution↗

PubMed2002

Decreased metallation and activity in subsets of mutant superoxide dismutases associated with familial amyotrophic lateral sclerosis.

Over 90 different mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) cause approximately 2% of amyotrophic lateral sclerosis (ALS) cases by an unknown mechanism. We engineered 14 different human ALS-related SOD1 mutants and obtained high yields of biologically metallated proteins from an Sf21 insect cell expression system. Both the wild type and mutant "as isolated" SOD1 variants were deficient in copper and were heterogeneous by native gel electrophoresis. By contrast, although three mutant SOD1s with substitutions near the metal binding sites (H46R, G85R, and D124V) were severely deficient in both copper and zinc ions, zinc deficiency was not a consistent feature shared by the as isolated mutants. Eight mutants (A4V, L38V, G41S, G72S, D76Y, D90A, G93A, and E133 Delta) exhibited normal SOD activity over pH 5.5-10.5, per equivalent of copper, consistent with the presumption that bound copper was in the proper metal-binding site and was fully active. The H48Q variant contained a high copper content yet was 100-fold less active than the wild type enzyme and exhibited a blue shift in the visible absorbance peak of bound Cu(II), indicating rearrangement of the Cu(II) coordination geometry. Further characterization of these as-isolated SOD1 proteins may provide new insights regarding mutant SOD1 enzyme toxicity in ALS.

Amino Acid Substitution↗

PubMed2002

Familial amyotrophic lateral sclerosis-associated mutations decrease the thermal stability of distinctly metallated species of human copper/zinc superoxide dismutase.

We report the thermal stability of wild type (WT) and 14 different variants of human copper/zinc superoxide dismutase (SOD1) associated with familial amyotrophic lateral sclerosis (FALS). Multiple endothermic unfolding transitions were observed by differential scanning calorimetry for partially metallated SOD1 enzymes isolated from a baculovirus system. We correlated the metal ion contents of SOD1 variants with the occurrence of distinct melting transitions. Altered thermal stability upon reduction of copper with dithionite identified transitions resulting from the unfolding of copper-containing SOD1 species. We demonstrated that copper or zinc binding to a subset of "WT-like" FALS mutants (A4V, L38V, G41S, G72S, D76Y, D90A, G93A, and E133Delta) conferred a similar degree of incremental stabilization as did metal ion binding to WT SOD1. However, these mutants were all destabilized by approximately 1-6 degrees C compared with the corresponding WT SOD1 species. Most of the "metal binding region" FALS mutants (H46R, G85R, D124V, D125H, and S134N) exhibited transitions that probably resulted from unfolding of metal-free species at approximately 4-12 degrees C below the observed melting of the least stable WT species. We conclude that decreased conformational stability shared by all of these mutant SOD1s may contribute to SOD1 toxicity in FALS.

Amino Acid Substitution↗