Search PubMed⌕ Search

Biomedical subjects

C H Williams

Publications and source records attributed to C H Williams.

At least 73 records · Page 4Linked to original sources
PubMed1994

Lactate monooxygenase. I. Expression of the mycobacterial gene in Escherichia coli and site-directed mutagenesis of lysine 266.

Lactate monooxygenase utilizes oxygen in the conversion of L-lactate to acetate, CO2, and water. The gene for the enzyme from Mycobacterium smegmatis had been cloned into Escherichia coli (Giegel, D. A., Williams, C. H., Jr., and Massey, V. (1990) J. Biol. Chem. 265, 6626-6632) and the derived amino acid sequence compared to glycolate oxidase and flavocytochrome b2, enzymes of known three-dimensional structure (Lindqvist, Y., and Brändén, C. I. (1989) J. Biol. Chem. 264, 3624-3628; Xia, Z. X., and Mathews, S. F. (1990) J. Mol. Biol. 212, 837-863). There is strong homology, especially around residues in the active site. The mechanism proposed for lactate monooxygenase involves an intermediate having a negative charge at the N(1)-position of the FMN. Based on the homology, lysine 266 is the residue suggested to neutralize that charge. Wild type enzyme and several forms of the enzyme altered at active site residues by site-directed mutagenesis have been expressed in E. coli and purification procedures developed. The properties determined for the recombinant wild type enzyme were, in every case, the same as those previously determined for the enzyme isolated from M. smegmatis. Mutation of lysine 266 to a methionine created K266M. The semiquinone showed spectral features different from those found in the wild type enzyme and was no longer thermodynamically stable. This indicates a redox potential for the enzyme-bound semiquinone/reduced flavin couple that is higher than the midpoint potential for the oxidized flavin/semiquinone couple. The two-electron redox potential was determined to be -180 mV at 25 degrees C, pH 7.0. In wild type enzyme, attack of the flavin ring by sulfite creates a negative charge at the FMN N(1)-position. In K266M, the stabilization of the sulfite adduct was 17,000-fold weaker (Kd approximately 10(-3) M) than in the wild type enzyme, with a rate of association that is lowered by 10,000-fold (kon = 1.2 M-1 s-1). The rate of reduction with L-lactate is significantly decreased in K266M. Unexpectedly, binding of substrate and inhibitors is significantly weaker in K266M than in the wild type enzyme. In all properties involving a negative charge at position N(1) of the FMN, K266M is distinctly different from wild type enzyme. This makes it quite likely that lysine 266 serves the postulated role of interacting with this negative charge.

Amino Acid Sequence↗
PubMed1994

Lactate monooxygenase. II. Site-directed mutagenesis of the postulated active site base histidine 290.

Lactate monooxygenase catalyzes the oxidation of L-lactate with molecular oxygen to acetate, CO2, and water. Histidine 290 has been proposed to be the active site base in lactate monooxygenase (Giegel, D. A., Williams, C. H., Jr., and Massey, V. (1990) J. Biol. Chem. 265, 6626-6632) and was mutated to a glutamine (H290Q). The mutant enzyme shows properties that support strongly the postulated function of the histidine. The ability of L-lactate to reduce the enzyme flavin is essentially abolished, whereas reoxidation of reduced enzyme with oxygen proceeds at 1.4 x 10(4) M-1 s-1, a rate essentially like that found in the wild type enzyme. The substrate, L-lactate, is bound with a Kd equal to 2.0 x 10(-2) M, and D-lactate, a competitive inhibitor with a Kd of 3.1 x 10(-3) M. Both values are similar to binding measured in the wild type enzyme. Unlike the situation with wild type enzyme, where the transition state analog oxalate is bound tightly in a two-step reaction involving proton uptake from solution (Ghisla, S., and Massey, V. (1977) J. Biol. Chem. 252, 6729-6735), the mutant enzyme binds oxalate weakly, in a single step reaction, with a Kd in the order of 0.1 M. No effect was observed upon varying the pH, indicating that binding does not include a protonation step. Replacing the histidine also has a significant effect on the ability of the enzyme to stabilize the flavin N(5)-sulfite adduct. Sulfite is bound at least 1000-fold weaker than it is in the wild type enzyme.

Amino Acid Sequence↗
PubMed1994

Lactate monooxygenase. III. Additive contributions of active site residues to catalytic efficiency and stabilization of an anionic transition state.

Lactate monooxygenase catalyzes the conversion of L-lactate to acetate, CO2, and water with incorporation of molecular oxygen. Several amino acid residues of lactate monooxygenase had been postulated to interact in specific ways with the bound substrate (Giegel, D. A., Williams, C. H., Jr., and Massey, V. (1990) J. Biol. Chem. 265, 6626-6632). Tyrosine 44 and arginine 293 were proposed to form a hydrogen bond and a salt bridge to the carboxyl-moiety of lactate. Tyrosine 152 was suggested to form a hydrogen bond to the alpha-hydroxyl group and could be involved in stabilizing a transient carbanionic intermediate of the substrate. The tyrosine residues were replaced with phenylalanines (Y44F, Y152F), and arginine 293 was mutated to a lysine (R293K). In all cases catalysis was significantly decreased; however, the binding affinity for L-lactate did not decrease. Instead, the Kd measured for Y152F was 10-fold lower than that for the wild type enzyme. The products of turnover with Y152F were similar to those with wild type enzyme, with 70-80% of the reaction proceeding to form acetate, CO2, and H2O. The catalytic reactions with both Y44F and R293K were substantially uncoupled, with between 60 and 80% of the catalytic turnover forming pyruvate and H2O2. For all mutant forms the reoxidation of enzyme with oxygen in the absence of pyruvate occurred at a rate similar to that measured for the wild type enzyme. The most important effect of the mutations was in the ability to stabilize the transition state analog oxalate. A linear relationship was found between the rate of reduction of the enzyme flavin and the dissociation constant for the binding of oxalate, demonstrating that many individual residues contribute to the lowering of the energy of the transition state, in addition to specific functions being assignable to some specific residues.

Amino Acid Sequence↗
PubMed1994

Mechanism of reduction of quinones by Trypanosoma congolense trypanothione reductase.

A number of quinones were analyzed as substrates for trypanothione reductase from Trypanosoma congolense, an enzyme responsible for the protection of trypanosomes against oxidative stress. Using NADPH as substrate, the maximal rate of the steady-state reaction at pH 7.5 was between 24 and 1.6 s-1 for 11 quinone substrates. The biomolecular steady-state rate constants for quinone reduction, V/Km, ranged from 240 to 1.9 x 10(5) M-1 s-1, and their logarithms exhibited a hyperbolic dependence on the one-electron-reduction potentials of the quinone substrate. The addition of NADP+ stimulated these rates, with V/Km values increasing with an increasing NADP+/NADPH ratio. The results of alkylation of the cysteine residue in the two-electron-reduced enzyme by iodoacetamide indicate that these residues are not primarily involved in the reduction of these quinones. Single-electron reduction of benzoquinone constitutes 40% of the total electron transfer from NADPH to quinone in the absence of NADP+, and increases to 80% at NADP+/NADPH ratios greater than 10. These steady-state results were confirmed in pre-steady-state rapid reaction experiments. The rate of reduced enzyme oxidation by 1,4-benzoquinone is approximately 100 times faster in the presence of NADP+ than in its absence. The reactivities of various pyridine nucleotide liganded forms of EH2 for quinone reduction are presumably affected by the electron density at FAD. We suggest that one-electron reduction of quinones occurs at a site distinct from the two active sites involved in hydride ion transfer and disulfide reduction.

Animals↗
PubMed1994

Crystal structure of Escherichia coli thioredoxin reductase refined at 2 A resolution. Implications for a large conformational change during catalysis.

The crystal structures of three forms of Escherichia coli thioredoxin reductase have been refined: the oxidized form of the wild-type enzyme at 2.1 A resolution, a variant containing a cysteine to serine mutation at the active site (Cys138Ser) at 2.0 A resolution, and a complex of this variant with nicotinamide adenine dinucleotide phosphate (NADP+) at 2.3 A resolution. The enzyme mechanism involves the transfer of reducing equivalents from reduced nicotinamide adenine dinucleotide phosphate (NADPH) to a disulfide bond in the enzyme, via a flavin adenine dinucleotide (FAD). Thioredoxin reductase contains FAD and NADPH binding domains that are structurally similar to the corresponding domains of the related enzyme glutathione reductase. The relative orientation of these domains is, however, very different in the two enzymes: when the FAD domains of thioredoxin and glutathione reductases are superimposed, the NADPH domain of one is rotated by 66 degrees with respect to the other. The observed binding mode of NADP+ in thioredoxin reductase is non-productive in that the nicotinamide ring is more than 17 A from the flavin ring system. While in glutathione reductase the redox active disulfide is located in the FAD domain, in thioredoxin reductase it is in the NADPH domain and is part of a four-residue sequence (Cys-Ala-Thr-Cys) that is close in structure to the corresponding region of thioredoxin (Cys-Gly-Pro-Cys), with a root-mean-square deviation of 0.22 A for atoms in the disulfide bonded ring. There are no significant conformational differences between the structure of the wild-type enzyme and that of the Cys138Ser mutant, except that a disulfide bond is not present in the latter. The disulfide bond is positioned productively in this conformation of the enzyme, i.e. it stacks against the flavin ring system in a position that would facilitate its reduction by the flavin. However, the cysteine residues are relatively inaccessible for interaction with the substrate, thioredoxin. These results suggest that thioredoxin reductase must undergo conformational changes during enzyme catalysis. All three structures reported here are for the same conformation of the enzyme and no direct evidence is available as yet for such conformational changes. The simplest possibility is that the NADPH domain rotates between the conformation observed here and an orientation similar to that seen in glutathione reductase. This would alternately place the nicotinamide ring and the disulfide bond near the flavin ring, and expose the cysteine residues for reaction with thioredoxin in the hypothetical conformation.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

Biological activity and receptor binding properties of some analogues of pQDPFLRFamide.

To investigate the role of the N-terminal region of the heptapeptide FMRFamide-like peptide, pQDPFLRFamide, three analogues were synthesized. The analogues [pQNPFLRFamide, pQDAibFLRFamide (Aib = aminoisobutyric acid) and pQDGFLRFamide] contained modifications at amino acid residues 2 and 3, which we believed might be critical for maintaining the bioactive conformation of the heptapeptide. The analogues were tested for their ability to bind to receptors in membranes from Helix aspersa circumoesophageal ganglia and for their biological effects on the isolated Helix heart, the Helix tentacle retractor muscle, and extensor-tibiae neuromuscular preparation of the locust. Schistocerca gregaria. The substitution of Asn for Asp2 and that of Aib for Pro3 were conservative with respect to retention of heptapeptide-like biological activity, whereas the substitution of Gly for Pro3 significantly improved the binding affinity of the peptide for the FMRFamide receptors and conferred on the peptide some characteristic FMRFamide-like biological activity. Thus, pQDPFLRFamide bioactivity may depend on a bent conformation in solution.

Amino Acid Sequence↗
PubMed1994

Biological activity and receptor binding properties of some C-terminally modified analogues of FMRFamide.

The functional role of the C-terminal amide group (-CONH2) of the molluscan regulatory peptide FMRFamide has been examined in two sets of analogues based on FnLKFamide and FnLRFamide (nL = norleucine). In each series the amide group was replaced by -CONHCH3, -CON(CH3)2, -CONHNH2, -COOCH3, -CH2OH, and -COOH. The analogues were tested for their ability to bind to receptors in membranes from Helix aspersa circumoesophageal ganglia and for their biological effects on the isolated Helix heart. The results indicate i) that agonist activity, but not binding to the receptor, requires the presence of the amide carbonyl group; ii) the hydrogen atoms of the amide group are not essential either for binding or for agonist activity (the mono- and dimethylamides were more effective than the parent compounds on both counts); iii) the is more effective an agonist than is the amide in stimulating Helix heart.

Animals↗
PubMed1993

Effects of the mutations Glu22 to Gln and Ala21 to Gly on the aggregation of a synthetic fragment of the Alzheimer's amyloid beta/A4 peptide.

We assessed the fibrillogenic properties of synthetic peptides corresponding to residues 13-26 of beta/A4 amyloid, containing either the normal sequence (beta 13 26) or the mutations Glu22 to Gln (beta 13-26Q22) and Ala21 to Gly (beta 13-26G21). The kinetics of aggregation were monitored at 37 degrees C and pH 7.4 by measuring the amount of peptide remaining in solution, using reverse-phase high performance liquid chromatography. Negative stain electron microscopy revealed that all of the peptides formed fibrils. However, beta 13-26Q22 showed greatly accelerated fibril formation compared to the other two. The results suggest that the Q22 mutation confers increased amyloidogenic properties on the beta/A4 peptide, whereas the G21 mutation acts by a different pathogenic mechanism.

Alanine↗
PubMed1993

Endopeptidase 3.4.24.11 converts N-1-(R,S)carboxy-3-phenylpropyl-Ala-Ala-Phe-p-carboxyanilide into a potent inhibitor of angiotensin-converting enzyme.

It was reported recently that N-1-(R,S)carboxy-3-phenylpropyl-Ala-Ala-Phe-p-carboxyanilide (CPP-A-A-F-pAB), an inhibitor of endopeptidase 3.4.24.15 (E-24.15), also inhibits angiotensin-converting enzyme (ACE) from rabbit lung. We have found that this compound is without effect on ACE purified from pig kidney, at a concentration some 1000-fold greater than the Ki reported for inhibition of the enzyme from lung. However, preincubation of CPP-A-A-F-pAB with neutral endopeptidase 3.4.24.11 (E-24.11) does result in potent inhibitory effects on ACE. We have shown this to be due to formation of a fragment, CPP-A-A, the structure of which is closely related to ACE inhibitors such as enalaprilat. CPP-A-A was found to be a potent inhibitor of pig ACE. Under the conditions used it had an IC50 value of 1.6 x 10(-8) M, compared with the value obtained for captopril of 7.5 x 10(-10) M. These results have important implications for studies of E-24.15 when using CPP-A-A-F-pAB in vivo or in crude tissue extracts where E-24.11 might also be present.

Amino Acid Sequence↗