Stereochemical course of the reaction catalyzed by the cyclic GMP phosphodiesterase from retinal rod outer segments.

F Eckstein,J W Karpen,J M Critchfield,L Stryer

doi:10.1016/s0021-9258(18)68187-6

Abstract

The stereochemical course of hydrolysis catalyzed by the cyclic GMP phosphodiesterase from bovine retinal rod outer segments was determined. The Sp diastereomer of guanosine 3',5'-cyclic monophosphorothioate was hydrolyzed by cyclic GMP phosphodiesterase in H2(18)O to give [16O,18O]guanosine 5'-monophosphorothioate. This isotopomer was reacted with diphenyl phosphorochloridate to form the two diastereomers of P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate. The 31P NMR spectrum of this mixture of diastereomers was identical to that obtained from [16O,18O]guanosine 5'-monophosphorothioate resulting from the hydrolysis of the Rp diastereomer of guanosine 5'-p-nitrophenyl phosphorothioate by snake venom phosphodiesterase. This finding indicates that the 18O is bridging in the Rp diastereomer of the P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate and nonbridging in the Sp diastereomer. As the snake venom phosphodiesterase reaction is known to proceed with retention of configuration, it follows that hydrolysis by retinal rod cyclic GMP phosphodiesterase proceeds with inversion of configuration at the phosphorus atom.

Highlights

The stereochemical course of hydrolysis catalyzed by the cyclic GMP phosphodiesterasefrom bovine retinal rod outer segments was determined
We report here the elucidation of the stereochemical course of the retinal rod cyclic GMP phosphodiesterase reaction
If H20 directly adds to bound cGMPin one step, the reaction is expected to proceed with inversion of configuration at the phosphorus atom

Summary

Introduction

The stereochemical course of hydrolysis catalyzed by the cyclic GMP phosphodiesterasefrom bovine retinal rod outer segments was determined. This isotopomer was reacted with diphenyl phosphorochloridate to form the two diastereomers of. As the snake venom phosphodiesterase reaction is known to proceed with retention of configuration, itfollows that hydrolysis by retinal rod cyclic GMP phosphodiesterase proceeds with inversion of configuration at the phosphorus atom. The cyclic GMP phosphodiesterase of retinal rod outer segments is a key enzyme in visual excitation (reviewed in Stryer, 1986; Liebman et al, 1987). We report here the elucidation of the stereochemical course of the retinal rod cyclic GMP phosphodiesterase reaction. This information is essential for unraveling the catalytic mechanism. A nucleophilic group on the enzyme first adds to cGMP to form a covalent intermediate, and H 2 0 thenattacks in-line to displace the enzyme, the overall reaction should proceed with retention of configuration

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