Stereochemical course of the reaction catalyzed by guanylate cyclase from bovine retinal rod outer segments.
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.
Koch
K W
KW
Department of Cell Biology, Sherman Fairchild Center, Stanford University School of Medicine, California 94305.
Eckstein
F
F
Stryer
L
L
D000818:Animals; D002417:Cattle; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D010786:Photoreceptor Cells / Q000201:enzymology*; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D006160:Guanosine Triphosphate; D006162:Guanylate Cyclase