Role of active-site residues Thr81, Ser82, Thr85, Gln157, and Tyr158 in yeast cystathionine beta-synthase catalysis and reaction specificity

Biochemistry. 2004 Feb 24;43(7):1963-71. doi: 10.1021/bi035496m.

Abstract

Cystathionine beta-synthase (CBS) effects the condensation of l-serine with l-homocysteine to form l-cystathionine. A series of active-site mutants, T81A, S82A, T85A, Q157A/E/H, and Y158F, was constructed to investigate effects on catalysis and reaction specificity in yeast CBS (yCBS). The effects of these mutations on the k(cat)/K(m)(L-Ser) for the beta-replacement reaction range from a reduction of only 3-fold for Y158F to below detectable levels for the Q157A and Q157E mutants. The order of importance of these residues to the beta-replacement reaction is Gln157 >or= Thr81 > Ser82 > Thr85 approximately Tyr158. All seven of the mutant enzymes catalyze a competing beta-elimination reaction, in which L-Ser is hydrolyzed to NH(3) and pyruvate. The ping-pong mechanism of CBS was thus expanded to include the latter reaction for these mutants. This activity is not detectable for wild-type yCBS, suggesting that the mutations result in a shift in the equilibrium between the open and the closed conformations of the active site of yCBS-substrate complexes. The Q157H and Y158F mutants additionally suffer suicide inhibition via a mechanism in which the released aminoacrylate intermediate covalently attacks the internal aldimine of the enzyme.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alanine / genetics
  • Amino Acid Substitution* / genetics
  • Binding Sites / genetics
  • Catalysis
  • Chromatography, Affinity
  • Cystathionine beta-Synthase / chemistry*
  • Cystathionine beta-Synthase / genetics
  • Glutamine / chemistry*
  • Glutamine / genetics
  • Kinetics
  • Models, Chemical
  • Mutagenesis, Site-Directed
  • Nickel
  • Phenylalanine / genetics
  • Pyruvic Acid / chemistry
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / genetics
  • Serine / chemistry*
  • Serine / genetics
  • Substrate Specificity / genetics
  • Threonine / chemistry*
  • Threonine / genetics
  • Tyrosine / chemistry*
  • Tyrosine / genetics

Substances

  • Saccharomyces cerevisiae Proteins
  • Glutamine
  • Threonine
  • Tyrosine
  • Serine
  • Phenylalanine
  • Nickel
  • Pyruvic Acid
  • Cystathionine beta-Synthase
  • Alanine