The Active-Site Arginine of S-Adenosylmethionine Synthetase Orients the Reaction Intermediate

Abstract

S-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 Pi 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 kcat reduced approximately 10(3)-fold, with a 7 to 10-fold increase in substrate Km values; the R244H mutant has an approximately 10(5)-fold decrease in kcat. In contrast, the kcat values for hydrolysis of added PPPi by the R244L 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 Pi 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 Pi 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.

Biochemical Basis for the Dominant Inheritance of Hypermethioninemia Associated with the R264H Mutation of theMAT1A Gene

“…AdoMet and PPP i are first synthesized from methionine and ATP; PPP i is subsequently hydrolyzed to PP i and P i to allow product release from the active site of the enzyme (2, 3). The function of the tripolyphosphatase activity of MAT is still under discussion (4,41). MAT activity of the R265H MAT I/III mutant was less than 1% of the activity of the WT enzyme, in agreement with previous data, which indicate that the active site of a dimeric MAT is configured by amino acid residues from both subunits (31,32,40).…”

“…Subsequently the tripolyphosphate generated is hydrolyzed to PP i and P i before the products of the reaction are released (2,3). The function of the tripolyphosphatase activity in the overall reaction catalyzed by MAT is still under discussion (4). In mammalian tissues three forms of MAT have been described that are the products of two distinct genes (5)(6)(7)(8).…”

“…Crystallographic studies of Escherichia coli and recombinant rat liver MAT show that the equivalent arginine 244 or 265, respectively, is located in the interface between the two subunits of the dimeric enzyme and is involved in a salt bridge with glutamic 42, or the homologous 58 in the rat enzyme, of the symmetric subunit, which contributes to the stabilization of the oligomeric state of MAT (31,32,40). Moreover, arginine 244 contributes to each active site and is located in the immediate vicinity of the polyphosphate group of ADP (4,31). It has been shown that replacement of arginine 264 by histidine in human MAT inactivates the enzyme.…”

“…Moreover, it has been proposed that this mutation hinders normal oligomeric formation (26). However, substitution of arginine 244 by leucine or histidine in E. coli MAT resulted in an inactive enzyme, which, in contrast, remains tetrameric with no apparent changes in the secondary structure (4). To understand the biochemical basis of the dominant inheritance of the phenotype associated with the R264H mutation of human MAT I/III, we have purified and characterized the homologous rat R265H MAT I/III mutant.…”

Enzymatic Properties of S-Adenosylmethionine Synthetase from the Archaeon Methanococcus jannaschii

“…AdoMet synthetase activity was determined by a [ 14 C]AdoMet cation exchange filter binding method (20). Assays were performed at 55°C in 25 mM Hepes⅐(CH 3 ) 4 N ϩ at pH 8.0 with 50 mM KCl, and 10 mM MgCl 2 .…”