Cytoplasmic guanylate cyclase, a heme-containing enzyme catalyses the conversion of guanosine 5'-trisphosphate to cyclic guanosine monophosphate which mediates numerous biochemical events including vascular smooth muscle relaxation, platelet deaggregation, photoreceptor cell signaling, ion transport in gastrointestinal cells, neurotransmission, relaxation of corpus cavernosum (impotence) and myeloid cell differentiation. The activity of soluble heme-containing guanylate cyclase is markedly increased by endothelium-derived relaxation factor (EDRF or nitric oxide) and several clinically employed nitric oxide generating vasodilators. Stroma free hemoglobin, considered by some as a possible blood substitute, binds nitric oxide strongly and thereby inhibits the activation of guanylate cyclase by nitric oxide. This proposal is directed towards the key area of understanding the mechanism of guanylate cyclase activation. The studies proposed here will define the role and the steric environment of heme and nitric oxide in guanylate cyclase activation by studying its reactions with various ligands in picosecond the second time domain. These studies will be made by single and double mixing stopped-flow spectrophotometry and ultrafast flash photolysis. The extensive data base for hemoglobins and other heme proteins will form the basis of interpreting the kinetic data for similar reactions of guanylate cyclase. The reactivity of the protein thiols in the activated and nonactivated forms the enzyme and their participation in the formation of nitrosothiol intermediates will also be investigated by kinetic methods. Studies on the reactivity of the heme-moiety and SH groups will be accompanied by the study of steady-state enzyme kinetics under identical conditions. We propose a model in which the dynamics of ligand binding with heme and its synergistic or antagonistic effect on heme binding with protein plays a crucial role in modulating the enzyme activity. We propose studies with heme-model compounds and heme-proteins to investigate the trigger mechanism in guanylate cyclase activation.
|Sucharov, Carmen; Bristow, Michael R; Port, J David (2008) miRNA expression in the failing human heart: functional correlates. J Mol Cell Cardiol 45:185-92|
|Kharitonov, V G; Russwurm, M; Magde, D et al. (1997) Dissociation of nitric oxide from soluble guanylate cyclase. Biochem Biophys Res Commun 239:284-6|
|Kharitonov, V G; Sharma, V S; Magde, D et al. (1997) Kinetics of nitric oxide dissociation from five- and six-coordinate nitrosyl hemes and heme proteins, including soluble guanylate cyclase. Biochemistry 36:6814-8|
|Kharitonov, V G; Sharma, V S; Pilz, R B et al. (1995) Basis of guanylate cyclase activation by carbon monoxide. Proc Natl Acad Sci U S A 92:2568-71|
|Kharitonov, V G; Sundquist, A R; Sharma, V S (1995) Kinetics of nitrosation of thiols by nitric oxide in the presence of oxygen. J Biol Chem 270:28158-64|
|Kharitonov, V G; Sundquist, A R; Sharma, V S (1994) Kinetics of nitric oxide autoxidation in aqueous solution. J Biol Chem 269:5881-3|