| Abstract
| - ATP:Co(I)rrinoid adenosyltransferase (ACA) catalyzes the conversion of cobalamin to coenzymeB12, an essential cofactor in animal metabolism. Several mutations of conserved residues in the active siteof human ACA have been identified in humans with methylmalonic aciduria. However, the catalytic roleof these residues remains unclear. To better understand the function of these residues and to determinehow the enzyme promotes catalysis, several variants of a human-type ACA from the lactic acid bacteriumLactobacillus reuteri (LrPduO) were kinetically and structurally characterized. Kinetic analyses of a seriesof alternate nucleotides were also performed. Substrate inhibition was observed at subsaturatingconcentrations of ATP, consistent with an ordered binding scheme where ATP is bound first by the enzyme.Modification or elimination of an active site, inter-subunit salt bridge resulted in a reduced “on” rate forATP binding, with a less significant disruption in the rate of subsequent catalytic steps. Kinetic and structuraldata demonstrate that residue Arg132 is not involved in orienting ATP in the active site but, rather, itstabilizes the altered substrate in the transition state. Two functional groups of ATP explain the reducedability of the enzyme to use alternate nucleotides: the amino group at the C-6 position of ATP contributes∼6 kcal/mol of free energy to ground state binding, and the nitrogen at the N-7 position assists incoordinating the magnesium ion in the active site. This study provides new insight into the role of substratebinding determinants and active site residues in the reaction catalyzed by a human-type ACA.
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