J. Biol. Chem., Vol. 265, Issue 17, 9659-9663, 06, 1990

Stereochemical course of the reaction catalyzed by guanylate cyclase from bovine retinal rod outer segments

KW Koch, F Eckstein and L Stryer
Department of Cell Biology, Sherman Fairchild Center, Stanford University School of Medicine, California 94305.

The stereochemical course of the reaction catalyzed by guanylate cyclase from bovine retinal rod outer segments was investigated using phosphorothioate analogs of GTP as chiral probes. (Sp)-Guanosine 5'-O- (1-thiotriphosphate) (Sp-GTP alpha S) is a substrate, whereas (Rp)-GTP alpha S is a competitive inhibitor (K1 = 0.1 mM), but not a substrate. (Sp)-GTP alpha S is converted into (Rp)-guanosine 3':5'- monophosphorothioate, showing that the reaction proceeds with inversion of configuration at the alpha-phosphorus atom. Km and Vmax for (Sp)-GTP alpha S (at low [Ca2+], 20 nM) are 3.7 mM and 1.1 nmol/min/mg of rhodopsin, respectively, compared with 1.1 mM and 23.1 nmol/min/mg of rhodopsin for GTP. Vmax for the cyclization of (Sp)-GTP alpha S, as for GTP, increases 10-20-fold when the calcium level is lowered. This activity change is centered at approximately 90 nM and has a Hill coefficient of 4.8. The configuration of the metal-substrate complex was determined by measuring the effectiveness of the Sp and Rp isomers of GTP alpha S and guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) in the presence of Mg2+ or Mn2+. (Sp)-GTP alpha S is a substrate with either Mg2+ or Mn2+, whereas (Rp)-GTP beta S is a substrate with only Mn2+. These findings suggest that the substrate is a metal-beta, gamma- bidentate complex with delta screwsense. We also found that the cyclization reaction catalyzed by the membrane-bound guanylate cyclase from sea urchin sperm proceeds with inversion of configuration at the alpha-phosphorus atom. The stereochemical course of the reactions catalyzed by all prokaryotic and eukaryotic adenylate cyclases and guanylate cyclases studied thus far is the same.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				J. H. Hurley Structure, Mechanism, and Regulation of Mammalian Adenylyl Cyclase J. Biol. Chem., March 19, 1999; 274(12): 7599 - 7602. [Full Text] [PDF]

				W.-J. Tang and J. H. Hurley Catalytic Mechanism and Regulation of Mammalian Adenylyl Cyclases Mol. Pharmacol., August 1, 1998; 54(2): 231 - 240. [Full Text]

				C. L. Tucker, J. H. Hurley, T. R. Miller, and J. B. Hurley Two amino acid substitutions convert a guanylyl cyclase, RetGC-1, into an adenylyl cyclase PNAS, May 26, 1998; 95(11): 5993 - 5997. [Abstract] [Full Text] [PDF]

				Y. Liu, A. E. Ruoho, V. D. Rao, and J. H. Hurley Catalytic mechanism of the adenylyl and guanylyl cyclases: Modeling and mutational analysis PNAS, December 9, 1997; 94(25): 13414 - 13419. [Abstract] [Full Text] [PDF]

				D. K. Thompson and D. L. Garbers Dominant Negative Mutations of the Guanylyl Cyclase-A Receptor J. Biol. Chem., January 6, 1995; 270(1): 425 - 430. [Abstract] [Full Text] [PDF]