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. GMP 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 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 ammonia transfer to this intermediate. Isolation and use of the adenylylated nucleotide intermediate enabled study of the amidotransfer 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 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.
GMP synthetase is the glutamine amidotransferase that catalyzes the final step in the guanylate branch of de novo purine biosynthesis. Conformational changes are required to efficiently couple distal active sites in the protein; however, the nature of these changes has remained elusive. Structural information derived from both limited proteolysis and sedimentation velocity experiments support the hypothesis of nucleotide-induced loop- and domain-closure in the protein. These results were combined with information from sequence conservation and precedents from other glutamine amidotransferases to develop the first structural model of GMPS in a closed, active state. In analyzing this Catalytic model, an interdomain salt bridge was identified residing in the same location as seen in other triad glutamine amidotransferases. Using mutagenesis and kinetic analysis, the salt bridge between H186 and E383 was shown to function as a connection between the two active sites. Mutations at these residues uncoupled the two half-reactions of the enzyme. The chemical events of nucleotide binding initiate a series of conformational changes that culminate in the establishment of a tunnel for ammonia as well as an activated glutaminase catalytic site. The results of this study provide a clearer understanding of the allostery of GMPS, where, for the first time, key substrate binding and interdomain contacts are modeled and analyzed.
Objective. To create and implement a class in ethnopharmacology that would educate student pharmacists on folk medicine, including home remedies and native plants that are used as alternative medicinal sources; active components of medicinal plants including toxicity issues and the mechanism of action of beneficial compounds, such as catechins and other flavonoids; and nutraceuticals and poisonous plants. Methods. In this three-credit hour class, herbal remedies are investigated from the standpoints of medical efficacy, potential toxicities and drug interactions with prescribed medications. Class discussions are conducted on the usefulness of remedies, the attitudes of practitioners toward traditional remedy use and the risks of relying on herbal preparations. Each student prepares a 15-minute presentation on a disease state, which covers modern pharmaceuticals and herbal or folk remedy alternatives used in that disease. Special emphasis is given to drug-herb interactions. Results. The class has gained popularity among students and consistently fills within the first hour of computerized registration. Students agree that being educated in the benefits and potential toxicities of herbal products will better prepare them to counsel their patients who use these remedies. The elective has been offered 10 times since 2007. Anecdotal comments from our alumni indicate that they have found the information to be very useful in their practice environments. Conclusion. Providing our students with a greater understanding of herbal remedies is essential to prepare them for practice. By including both the uses and potential toxicities, the student pharmacist is able to counsel her patients from a standpoint of expertise on these self-administered remedies.
The enzyme N1-(5'-phosphoribosyl) adenosine-5'-monophosphate cyclohydrolase (PR-AMP cyclohydrolase) is a Zn2+ metalloprotein encoded by the hisI gene. It catalyzes the third step of histidine biosynthesis (Scheme 1), an uncommon ring opening of a purine heterocycle for use in primary metabolism. A three dimensional structure of the enzyme from Methanobacterium thermoautotrophicum has revealed that three conserved cysteine residues occur at the dimer interface and likely form the catalytic site. To investigate of the functions of these cysteines in the enzyme from Methanococcus vannielii, a series of biochemical studies were pursued to test the basic hypothesis regarding their roles in catalysis. Inactivation of the enzyme activity by methyl methane thiosulfonate (MMTS) or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) also compromised the Zn2+ binding properties of the protein inducing loss of up to 90% of the metal. Overall reaction stoichiometry and the potassium cyanide (KCN) induced cleavage of the protein suggested that all three cysteines were modified in the process. The enzyme was protected from DTNB-induced inactivation by inclusion of the substrate PR-AMP while Mg2+, a metal required for catalytic activity, enhanced the rate of inactivation. Site directed mutations of the conserved C93, C109, C116 and the double mutant C109/C116 were prepared and analyzed for catalytic activity, Zn2+ content, and reactivity with DTNB. Substitution of alanine for each of the conserved cysteines showed no measurable catalytic activity and only the C116A was still capable of binding Zn2+. Reactions of DTNB with the C109A/C116A double mutant showed that C93 is completely modified within 0.5 s. A model consistent with these data involves a DTNB-induced mixed disulfide linkage between C93 and C109 or C116, followed by ejection of the active site Zn2+ and provides further evidence that the Zn2+ coordination site involves the three conserved cysteine residues. The C93 reactivity is modulated by the presence of the Zn2+ and Mg2+and substantiates the role of this residue as a metal ligand. In addition, Mg2+ ligand binding site(s) indicated by the structural analysis were probed by site-directed mutagenesis of three key aspartate residues flanking the conserved C93 which were shown to have a functional impact on catalysis, cysteine activation, and metal (zinc) binding capacity. The unique amino acid sequence, the dynamic properties of the cysteine ligands involved in Zn2+ coordination and the requirement for a second metal (Mg2+) are discussed in the context of their roles in catalysis. The results are consistent with a Zn+2 mediated activation of H2O mechanism involving histidine as a general base that has features similar to but distinct from those of previously characterized purine and pyrimidine deaminases.
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