Helen M. Wilks
University of Bristol
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Featured researches published by Helen M. Wilks.
Biochemical and Biophysical Research Communications | 1987
Anthony R. Clarke; Corinne J. Smith; Keith W. Hart; Helen M. Wilks; William N. Chia; Thomas V. Lee; Jens J. Birktoft; Leonard J. Banaszak; David A. Barstow; Tony Atkinson; J. John Holbrook
Using site-directed mutagenesis on the lactate dehydrogenase gene from Bacillus stearothermophilus, three amino acid substitutions have been made at sites in the enzyme which we suggest in part determine specificity toward different hydroxyacids (R-CHOH-COOH). To change the preferred substrates from the pyruvate/lactate pair (R = -CH3) to the oxaloacetate/malate pair (R = -CH2-COO-), the volume of the active site was increased (thr 246----gly), an acid was neutralized (asp-197----asn) and a base was introduced (gln-102 - greater than arg). The wild type enzyme has a catalytic specificity for pyruvate over oxaloacetate of 1000 whereas the triple mutant has a specificity for oxaloacetate over pyruvate of 500. Despite the severity and extent of these active site alterations, the malate dehydrogenase so produced retains a reasonably fast catalytic rate constant (20 s-1 for oxaloacetate reduction) and is still allosterically controlled by fructose-1,6-bisphosphate.
Biochemical and Biophysical Research Communications | 1989
Daniel Bur; Tony Clarke; James D. Friesen; Marvin Gold; Keith W. Hart; J. John Holbrook; J. Bryan Jones; Marcel A. Luyten; Helen M. Wilks
The function of the amino acid Thr246 in L-lactate dehydrogenase from Bacillus stearothermophilus has been investigated by site-directed replacement with glycine. Kinetic experiments with a number of 2-oxo acids showed strongly reduced activity for the mutated enzyme. However, the mutant enzyme shows a relative preference for the large hydrophobic sidechains of alpha-keto acids and an even higher specific activity than the wild-type lactate dehydrogenase for the polar oxaloacetate substrate. Graphic analyses indicate that the loss of one hydrogen bond, or intrusion of water into the active site, might be responsible for the reduced activity. The kinetic results suggest that the binding modes of bulky hydrophobic or polar substrates compensate to some degree for the partially disrupted active site.
Current Opinion in Biotechnology | 1991
Helen M. Wilks; J. John Holbrook
New substrate specificities can be introduced into existing enzymes for the purpose of making them more suitable for the chemoenzymic synthesis of single compound drugs and other chiral compounds. The most productive route used in the past year has involved the utilization of the catalytic and substrate-binding properties from homologous enzymes found in nature, one example being the broadening of the substrate specificity of yeast alcohol dehydrogenase. Other highlights include the creation of thermostable dehydrogenases that will interconvert NADPH and NADH, and the design of mutant enzymes with improved catalytic rates compared with their wild-type counterparts.
Science | 1988
Helen M. Wilks; Keith W. Hart; Raymond Feeney; Cameron R. Dunn; Hilary Muirhead; William Chia; David A. Barstow; Tony Atkinson; Anthony R. Clarke; J. John Holbrook
Philosophical Transactions of the Royal Society B | 1991
Cameron R. Dunn; Helen M. Wilks; D. J. Halsall; Tony Atkinson; Anthony R. Clarke; Hilary Muirhead; J. John Holbrook
Biochemistry | 1990
Helen M. Wilks; David J. Halsall; Tony Atkinson; William Chia; Anthony R. Clarke; J. John Holbrook
Biochemistry | 1988
Anthony R. Clarke; Helen M. Wilks; David A. Barstow; Tony Atkinson; William Chia; J. John Holbrook
Biochemistry | 1992
Helen M. Wilks; Kathleen M. Moreton; David J. Halsall; Keith W. Hart; Richard D. Sessions; Anthony R. Clarke; J. John Holbrook
Biochemistry | 1992
Rm Jackson; Jl Gelpi; Dc Emery; Helen M. Wilks; Km Moreton; David J. Halsall; Rn Sleigh; M Behan-Martin; Gr Jones; Anthony R. Clarke; J. John Holbrook
Biochemical Society Transactions | 1987
Tony Atkinson; David A. Barstow; William N. Chia; Anthony R. Clarke; Keith W. Hart; Adam D.B. Waldman; Dale B. Wigley; Helen M. Wilks; J. John Holbrook