Contribution of a conserved arginine near the active site of Escherichia coli D-serine dehydratase to cofactor affinity and catalytic activity.
We have employed site-directed mutagenesis to investigate the contribution of a conserved arginyl residue to the catalytic activity and cofactor affinity of D-serine dehydratase, a model pyridoxal 5'-phosphate (vitamin B6) enzyme. Replacement of R-120 in the active site peptide of D-serine dehydratase by L decreased the affinity of the enzyme for pyridoxal 5'-phosphate by 20-fold and reduced turnover by 5-8-fold. kappa cat displayed modified substrate alpha-deuterium isotope effects and altered dependence on both temperature and pH. Analysis of the pH rate profiles of DSD and the R-120----L variant indicated that R-120 interacts electrostatically with catalytically essential ionizable groups at the active site of wild type D-serine dehydratase. The decrease in cofactor affinity observed for DSD(R120L) was not accompanied by significant perturbations in the UV, CD, or 31P NMR spectrum of the holoenzyme, suggesting that the contribution of R-120 to pyridoxal phosphate affinity may be indirect or else involve an interaction with a cofactor functional group other than the 5'-phosphoryl moiety. The properties of two other site-directed variants of D-serine dehydratase indicated that the pyridoxal 5'-phosphate:K-118 Schiff base was indifferent to a small change in the shape of the side chain at position 117 (I-117----L), whereas replacement of K-118 by H resulted in undetectable levels of enzyme. A poor ability to bind cofactor may have rendered DSD(K118H) susceptible to intracellular proteolysis.
Marceau
M
M
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
Lewis
S D
SD
Kojiro
C L
CL
Shafer
J A
JA
D000595:Amino Acid Sequence; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D004274:DNA, Recombinant / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D012695:L-Serine Dehydratase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000378:metabolism*
D004274:DNA, Recombinant; D011732:Pyridoxal Phosphate; D001120:Arginine; D012695:L-Serine Dehydratase