Abstract
Glutamine amidotransferases catalyze the amination of a wide range of molecules using the amide nitrogen of glutamine. The family provides numerous examples for study of multi-active-site regulation and interdomain communication in proteins. Guanosine 5'-monophosphate synthetase (GMPS) is one of three glutamine amidotransferases in de novo purine biosynthesis and is responsible for the last step in the guanosine branch of the pathway, the amination of xanthosine 5'-monophosphate (XMP). In several amidotransferases, the intramolecular path of ammonia from glutamine to substrate is understood; however, the crystal structure of GMPS only hinted at the details of such transfer. Rapid kinetics studies provide insight into the mechanism of the substrate-induced changes in this complex enzyme. Rapid mixing of GMPS with substrates also manifests absorbance changes that report on the kinetics of formation of a reactive intermediate as well as steps in the process of rapid transfer of ammonia to this intermediate. Isolation and use of the adenylylated nucleotide intermediate allowed the study of the amido transfer reaction distinct from the ATP-dependent reaction. Changes in intrinsic tryptophan fluorescence upon mixing of enzyme with XMP suggest a conformational change upon substrate binding, likely the ordering of a highly conserved loop in addition to global domain motions. In the GMPS reaction, all forward rates before product release appear to be faster than steady-state turnover, implying that release is likely rate-limiting. These studies establish the functional role of a substrate-induced conformational change in the GMPS catalytic cycle and provide a kinetic context for the formation of an ammonia channel linking the distinct active sites.