Replacement of Asp333 with Asn by site-directed mutagenesis changes the substrate specificity of Escherichia coli adenylosuccinate synthetase from guanosine 5'-triphosphate to xanthosine 5'-triphosphate.
The aspartate residue of the (N/T)KXD concensus sequence for GTP-binding proteins is present in the eight available sequences of adenylosuccinate synthetase. Reported here is a comprehensive analysis of the substrate specificity of mutant enzymes, where the conserved Asp333 of the synthetase from Escherichia coli is changed to asparagine, glutamate, and glutamine by site-directed mutagenesis. The mutants D333N, D333E, and D333Q generally show decreased kcat values and increased Km values for GTP. The decreased values of kcat exhibited by the mutants indicate that the interactions between Asp333 and the guanine are relayed by some mechanism to the catalytic residues around the gamma-phosphate of GTP, and that the energy provided by the interaction between Asp333 and the guanine moiety of GTP is utilized for rearrangement of the catalytic residues. The three mutants each have higher affinity for xanthosine 5'-triphosphate (XTP) and ITP than does the wild-type enzyme. In fact, the D333N mutant uses XTP more effectively than the wild-type enzyme employs GTP as a substrate. The side-chain of Asp333 forms hydrogen bonds with the N-1 and the exocyclic amino group of the guanine base of GTP. In the D333N mutant, this interaction is probably replaced by hydrogen bonds between the amide side chain of Asn333 and N-1 and the 2-oxo group of XTP. The D333Q mutant can use UTP as a substrate more effectively than the wild-type enzyme. The longer side chain of glutamine at residue 333 favors pyrimidine nucleotides over the purine nucleotides, GTP, XTP, and ITP. These results demonstrate that Asp333 in the (N/T)KXD consensus sequence of adenylosuccinate synthetase from E. coli is a determinant for GTP-specificity.
Kang
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Department of Biochemistry and Biophysics, Iowa State University, Ames 50010.
Sun
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Honzatko
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Fromm
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HJ
D000265:Adenylosuccinate Synthase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001216:Asparagine / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism*; D001483:Base Sequence; D002942:Circular Dichroism; D018076:DNA, Complementary; D004926:Escherichia coli / Q000201:enzymology*; D019204:GTP-Binding Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012265:Ribonucleotides / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics
D018076:DNA, Complementary; D012265:Ribonucleotides; D001224:Aspartic Acid; C065326:xanthosine 5'-triphosphate; D001216:Asparagine; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D000265:Adenylosuccinate Synthase