Kinetic evidence for the formation of D-alanyl phosphate in the mechanism of D-alanyl-D-alanine ligase.
The steady state kinetic mechanism, molecular isotope exchange and the positional isotope exchange (PIX) reactions of D-alanyl-D-alanine ligase from Salmonella typhimurium have been studied. The kinetic mechanism has been determined to be ordered Ter-Ter from initial velocity and product inhibition experiments. The first substrate to bind is ATP followed by the addition of 2 mol of D-alanine. Pi is released, and then D-alanyl-D-alanine and ADP dissociate from the enzyme surface. In the reverse direction D-alanyl-D-alanine exhibits complete substrate inhibition (Ki = 1.15 +/- 0.05 mM) by binding to the enzyme-ATP complex. In the presence of D-alanine, D-alanyl-D-alanine ligase catalyzed the positional exchange of the beta,gamma-bridge oxygen in [gamma-18O4]ATP to a beta-nonbridge position. Two possible alternate dead-end substrate analogs, D-2-chloropropionic acid and isobutyric acid, did not induce a positional isotope exchange in [gamma-18O4]ATP. The positional isotope exchange rate is diminished relative to the net substrate turnover as the concentration of D-alanine is increased. This is consistent with the ordered Ter-Ter mechanism as determined by the steady state kinetic experiments. The ratio of the positional isotope exchange rate relative to the net chemical turnover of substrate (Vex/Vchem) approaches a value of 1.4 as the concentration of D-alanine becomes very small. This ratio is 100 times larger than the ratio of the maximal reverse and forward chemical reaction velocities (V2/V1). This situation is only possible when the reaction mechanism proceeds in two distinct steps and the first step is much faster than the second step. The enzyme was also found to catalyze the molecular isotope exchange of radiolabeled D-alanine with D-alanyl-D-alanine in the presence of phosphate. These results are consistent with the formation of D-alanyl phosphate as a kinetically competent intermediate.
Mullins
L S
LS
Department of Chemistry, Texas A & M University, College Station 77843.
Zawadzke
L E
LE
Walsh
C T
CT
Raushel
F M
FM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000031:analogs & derivatives* / Q000378:metabolism; D000814:Aniline Compounds; D003545:Cysteine / Q000031:analogs & derivatives / Q000032:analysis; D007700:Kinetics; D010103:Oxygen Isotopes; D010453:Peptide Synthases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation; D012486:Salmonella typhimurium / Q000201:enzymology*; D013438:Sulfhydryl Compounds
D000814:Aniline Compounds; D010103:Oxygen Isotopes; D010710:Phosphates; D013438:Sulfhydryl Compounds; C064315:alanylphosphate; C064317:S-(4-aminophenyl)cysteine; D000255:Adenosine Triphosphate; C064316:4-aminothiophenol; D010453:Peptide Synthases; C019638:D-alanylalanine synthetase; D003545:Cysteine; D000409:Alanine