catalytic Tyr-Glu dyad is demonstrated by site-directed mutagenesis and kinetic experiments that compare the wild-type enzymes to their respective mutant proteins using pHârate profiles, 32P-incorporation experiments, solvent isotope effects, proton inventory, and viscosity variation
the kinetic mechanism is bi-ter where ATP is the first substrate to bind followed by (2S,5S)-5-carboxymethyl proline and diphosphate is the last product released. Low activity with (2S,5R)-5-carboxymethylproline or (2R,5R)-5-carboxymethylproline
the uncompetitive nature of the inhibition of the dead-end inhibitor, L-proline, versus ATP is consistent with a mechanism with ordered substrate binding where ATP binds first followed by (2S,5S)-carboxymethylproline
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sitting drop method, the crystal structures of apo-CarA and CarA complexed with the substrate (2S,5S)-5-carboxymethylproline, ATP analog alpha,beta-methyleneadenosine 5'-triphosphate, and a single Mg2+
mutant exhibits a 5fold increase in Km of (2S,5S)-5-carboxymethylproline and a 165fold decrease in specificity constant compared to wild-type enzyme. kcat/Km is 165fold lower compared to wild-type value
retains a functional general base with a pKa of about 7.4 for kcat. The acidic region of the log (kcat/Km) versus pH profiles displays an ionizable group with a pKa of about 7.7. kcat/Km is 1.4fold lower compared to wild-type value