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Reczkowski RS , Taylor JC , Markham GD
The active-site arginine of S-adenosylmethionine synthetase orients the reaction intermediate
Biochemistry. 1998 Sep 29;37(39) :13499-13506
AbstractS-Adenosylmethionine (AdoMet) synthetase catalyzes the formation of AdoMet and tripolyphosphate (PPPi) from ATP and L- methionine and the subsequent hydrolysis of the PPPi to PPi and P-i before product release. Little is known about the roles of active-site residues involved in catalysis of the two sequential reactions that occur at opposite ends of the polyphosphate chain. Crystallographic studies of Escherichia coli AdoMet synthetase showed that arginine-244 is the only arginine near the polyphosphate-binding site. Arginine-244 is embedded as the seventh residue in the conserved sequence DxGxTxxKxI which is also found at the active site of inorganic pyrophosphatases, suggesting a potential pyrophosphate-binding motif, Chemical modification of AdoMet synthetase by the arginine-specific reagents phenylglyoxal or p- hydroxyphenylglyoxal inactivates the enzyme. ATP and PPPi protect the enzyme from inactivation, consistent with the presence of an important arginine residue in the vicinity of the polyphosphate-binding site. Site-specific mutagenesis has been used to change the conserved arginine-244 to either leucine (R244L) or histidine (R244H). In the overall reaction, the R244L mutant has the k(cat) reduced similar to 10(3)-fold, with a 7 to 10-fold increase in substrate K-m values; the R244H mutant has an similar to 10(5)-fold decrease in k(cat). In contrast, the k(cat) values for hydrolysis of added PPPi by the K244L and R244H mutants have been reduced by less than 2 orders of magnitude. In contrast to the wild-type enzyme in which 98% of the P-i formed originates as the gamma-phosphoryl group of ATP, in the R244L mutant the orientation of the PPPi intermediate equilibrates at the active site yielding equal amounts of P-i from the alpha- and gamma-phosphoryl groups of ATP. Thus, the active-site arginine has a profound role in the cleavage of PPPi from ATP during AdoMet formation and in maintaining the orientation of PPPi in the active site, while playing a lesser role in the subsequent PPPi hydrolytic reaction.
NotesTimes Cited: 10 English Article 127UA BIOCHEMISTRY-USA