The long range objective of the proposed study is to learn detailed mechanism by which enzyme-catalyzed nucleophilic substitution occurs at phosphorus and to clearly define the structural requirements for the formation of a functional enzyme-substrate(s) complex by studies with phosphoglycerate kinase (PGK). PGK, likely catalyzes the reaction via a hinge-bending motion of two domains, which results in the closure of an active site cleft, as suggested by x-ray studies. However, mechanism of the enzyme is not known at a chemical and structural level. This proposal will examine the mechanism of catalysis. The active site structure and its relationship to catalysis with PGK by studying the conformations and the arrangements of he substrates and substrate analogs, the coordination number and the position of the activator metal ion with respect to substrates at the active site of PGK. The identities and the roles of the amino acid residues near the substrates will be determined and their qualitative and quantitative contributions to catalysis will be studied. These goals will be achieved by focusing on 5 specific aims: 1) The arrangement and conformation of substrates at the active site will be determined by measuring the distances to substrate nuclei from a paramagnetic reference point, such as an activator metal ion Mn2+ or Co2+ or a paramagnetic substrate analog Cr(III) ATP. 2) Intramolecular nuclear Overhauser effect studies (NOE) will be performed to define the sugar pucker and glycosidic torsional angle (chi) of the nucleotide at the active site. Combined use of the data in aims 1 and 2 will provide more precise description of the conformation of ATP (or AMPP-CH2-P) in the different complexes of enzyme, metal, ATP and 3-PGA. 3) Detailed knowledge of structure of metal AMPP-CH2-P-3-PGA will be completed by determining coordination number of the metal ion by NMR and Electron Spin Echo Modulation Studies in the various complexes of enzyme, metal, AMPP-CH2-P and 3-PGA. 4) The identities of amino acid residues near substrate protons will be determined by intermolecular NOE studies in the diamagnetic complexes of enzyme, Mg2+, AMPP-CH2-P and 3-PGA (see the preliminary results). The distances from paramagnetic metal ion to these amino acids will be determined in separate experiments by using totally deuterated enzyme with only the designated amino acid in protonated form to yield visible NMR spectrum. 5) The roles of catalytically important amino acids in catalysis (determined in 4) will be tested by site specific mutations at these positions. These mutants will also be studied in detail by using the same nuclear magnetic resonance methods mentioned above. The proposed studies are both necessary and fundamental to our understanding of the catalysis by phosphoglycerate kinase and the results of these studies may provide useful information for the studies with the other kinases, which catalyze reactions by a similar hinge-bending action. This approach may be useful to elucidate general principles of enzyme chemistry, and to develop and critically compare various spectroscopic approaches to enzyme structure and mechanism.
