STRUCTURE/FUNCTION RELATIONSHIPS OF GUANYLATE KINASE

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Guanylate kinase (GK) plays an essential role in the cGMP cycle and may be involved in guanine nucleotide-mediated signal transduction pathways. The long-term goal of the project is to establish quantitative structure- function relationships for yeast GK that will account for its catalytic mechanism and nucleotide specificity, using a combination of kinetic and thermodynamic methods, site-directed mutagenesis, and biophysical methods. The specific aims of the project include: (1) To determine the kinetic pathway and energetics of the GK-catalyzed reaction by steady- state and transient kinetics and thermodynamic measurements. The goal is to obtain a complete free energy profile which will be the basis for dissecting the contributions of individual amino acid residues to catalysis and substrate specificity. (2) To evaluate the roles of the active site residues in catalysis and nucleotide specificity by site- directed mutagenesis. The contributions of these residues to each step of catalysis will be quantitated by evaluating the effects of mutations on the reaction profile as described in Specific Aim 1. Furthermore, how the amino acid residues interact with the substrates will be probed by examining the kinetics and/or stereochemistry of substrate analogues. (3) To assess the effects of mutations on the structure and stability by biophysical methods. The goal is not only to define the roles of the amino acid residues in structure and conformational stability but also to provide the essential structural information for quantitative interpretation of the results obtained in Specific Aim 2. (4) To identify the amino acid residues in close proximity to the bound ATP by NMR and dock the nucleotide into the crystal structure of yeast GK by computer graphics with the distance constraints from the NMR data. (5) To engineer new substrate specificity. The initial goal is to redesign the GMP site by mutagenesis so that the enzyme catalyzes phosphoryl transfer from ATP specifically to IMP, XMP, or AMP. The ATP site will also be modified at the late stage of the project so that it is specific for GTP, ITP, or XTP.