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The active-site arginine of S-adenosylmethionine synthetase orients the reaction intermediate
R S Reczkowski1, J C Taylor, G D Markham
1Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
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.
Insights
The active-site arginine in S-Adenosylmethionine synthetase is crucial for cleaving tripolyphosphate (PPPi) from ATP and orienting the PPPi intermediate. Mutation of this arginine significantly impairs overall catalysis but has a lesser effect on subsequent PPPi hydrolysis.
Area of Science:
- Enzymology
- Biochemistry
- Structural Biology
Background:
- S-Adenosylmethionine (AdoMet) synthetase catalyzes ATP and L-methionine into AdoMet and tripolyphosphate (PPPi).
- The roles of active-site residues in the sequential reactions of AdoMet synthetase are not well understood.
- Arginine-244 (R244) is identified as a key residue near the polyphosphate-binding site, sharing a conserved motif with inorganic pyrophosphatases.
Purpose of the Study:
- To investigate the role of arginine-244 in the catalytic mechanism of S-Adenosylmethionine synthetase.
- To elucidate the function of R244 in both the cleavage of tripolyphosphate (PPPi) from ATP and its subsequent hydrolysis.
Main Methods:
- Chemical modification of AdoMet synthetase using arginine-specific reagents (phenylglyoxal, p-hydroxyphenylglyoxal).
- Site-specific mutagenesis to generate R244L and R244H mutants.
- Enzyme kinetics assays to determine catalytic rates (kcat) and substrate affinities (Km) for wild-type and mutant enzymes.
- Analysis of inorganic phosphate (Pi) origin to assess PPPi intermediate orientation.
Main Results:
- Arginine-specific modification inactivated the enzyme, with protection offered by ATP and PPPi, confirming R244's importance.
- R244L and R244H mutants exhibited significantly reduced overall catalytic efficiency (kcat decreased by 10^3- and 10^5-fold, respectively).
- Mutations had a less pronounced effect on the hydrolysis of added PPPi (kcat reduced by less than 2 orders of magnitude).
- The R244L mutant showed altered orientation of the PPPi intermediate, leading to equal Pi formation from alpha- and gamma-phosphoryl groups of ATP, unlike the wild-type.
Conclusions:
- The active-site arginine (R244) plays a critical role in the cleavage of PPPi from ATP during AdoMet formation.
- R244 is essential for maintaining the correct orientation of the PPPi intermediate within the active site.
- While important for overall catalysis, R244 has a less significant role in the subsequent hydrolysis of PPPi.