Effects of temperature on allosteric and catalytic properties of the cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver.

H Wada,J.C Osborne,V.C Manganiello

doi:10.1016/s0021-9258(18)61166-4

Abstract

We have investigated effects of temperature on the catalytic and allosteric properties of the cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver. Vmax for cAMP and cGMP increased as assay temperature increased from 5 to 45 degrees C. At substrate concentrations below Kmapp, however, hydrolysis increased as temperature decreased from 45 to 5 degrees C and was much greater at 5 degrees C than at 45 degrees C. As assay temperature decreased, Kmapp for cAMP and cGMP decreased. Hill coefficients for cAMP and cGMP were approximately 1.9 at 45 degrees C and 1.2-1.0 at 5 degrees C. cGMP stimulated hydrolysis of 0.5 microM [3H]cAMP at all assay temperatures. Although maximal activity stimulated by cGMP, like Vmax, was lowest at 5 degrees C, presumably because of the effect of temperature on catalytic activity, the apparent activation constant (K alpha app) for cGMP stimulation was lower at 5 degrees C than at 45 degrees C. Thus, affinity for both substrate and effector was increased at 5 degrees C, suggesting that low temperature promotes transitions of the cGMP-stimulated phosphodiesterase to a "high affinity" state. That cGMP stimulated cAMP hydrolysis at 5 degrees C suggests that temperature-induced transitions are incomplete and/or readily reversible. In assays at 30 degrees C competitive inhibitors, like substrates, induce allosteric transitions which result in enhanced hydrolysis of low substrate (1.0 microM [3H] cAMP) concentrations. At higher substrate concentrations (50 microM [3H]cAMP), with the enzyme in the "activated" state, inhibitors compete with substrate at catalytic sites and reduce hydrolysis. At 45 degrees C, as at 30 degrees C, 1-methyl-3-isobutylxanthine (IBMX) and papaverine increased hydrolysis of 1.0 microM [3H]cAMP and reduced hydrolysis of 50 microM [3H]cAMP. At 5 degrees C, however, IBMX and papaverine inhibited hydrolysis of both 1.0 and 50 microM [3H]cAMP. Enzyme activity was relatively more sensitive to inhibition by IBMX at 5 degrees C than at 45 degrees C. Taken together, these observations support the notion that low temperature induces incomplete or readily reversible transitions to the high affinity state for substrates, effectors, and inhibitors. These observed effects of temperature also point out that enzyme determinants and topographical features responsible for transitions to the high affinity state and expression of catalytic activity can be regulated independently.

Highlights

We have investigated effectsof temperature on the The cGMP-stimulatedcyclic nucleotidephosphodiesterase, oatalytic and allosteric propertiesof the cGMP-stimu- purified from bovine heart, adrenal, and liver [1,2,3], hydrolated cyclic nucleotide phosphodiesterase from calf lyzes both cAMP and cGMP with positively cooperative kiliver
Enzyme activity was relatively and 1 at 5 "C. From these and other observations and from effectsof competitiveinhibitors on cAMP hydrolysisin assays at different temperatures, we suggest that low temperature promotes transitions of the c G M P - stimula ~phosphodiesterase to the high affinity state and higher temperatures to the low affinity state
In assays carried out at higher substrate concentrations, i.e. 50 p~ [3H]cAMP,hydrolysis was inhibited by both IBMX and papaverine at 5, 30, and 45 "C, the IC5, being highest at 45 "C (Fig. 6)

Summary

THEJOURNALOF BIOLOGICACLHEMISTRY

5139-5144,1987 Printed in U.S.A. Effects of Temperature onAllosteric and CatalyticProperties of the cGMP-stimulated Cyclic Nucleotide Phosphodiesterase from Calf Liver*. We have investigated effectsof temperature on the The cGMP-stimulatedcyclic nucleotidephosphodiesterase, oatalytic and allosteric propertiesof the cGMP-stimu- purified from bovine heart, adrenal, and liver [1,2,3], hydrolated cyclic nucleotide phosphodiesterase from calf lyzes both cAMP and cGMP with positively cooperative kiliver. Like substrates, bind to thelow affinity parent activation constan(tKim)for cGMP stimulation form of the phosphodiesterase, induce allosteric transitions, was lowerat 5 "Cthan at 45 OC.affinity forboth and increase hydrolysis of low concentrations of substrate; at substrate and effector was increaseadt 5 "C, suggest- higher substrate concentrations (ie.with the activated ening that low temperature promotes transitions of the zyme), these compounds competewith substrate at catalytic cGMP-stimulated phosphodiesterase to a "high affin- sites [5, 6].

Whereas Vmaxfor cAMP and cGMP hydrolysis increases as

RESULTS

Findings

DISCUSSION