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Evidence for essential histidine and cysteine residues in calcium/calmodulin-sensitive cyclic nucleotide phosphodiesterase.

Authors
Type
Published Article
Journal
Biochemistry
Publication Date
Volume
30
Issue
27
Pages
6754–6760
Identifiers
PMID: 1648392
Source
Medline
License
Unknown

Abstract

Ca/calmodulin-sensitive cyclic nucleotide phosphodiesterase (CaM-PDE) is an important enzyme regulating cGMP levels and relaxation of vascular smooth muscle. This modification study was conducted mostly with bovine brain CaM-PDE to identify essential functional groups involved in catalysis. The effect of pH on Vmax/Km indicates two essential residues with pKa values of 6.4 and 8.2. Diethyl pyrocarbonate (DEP), a histidine-modifying agent, inhibits CaM-PDE with a second-order rate constant of 130 M-1 min-1 at pH 7.0 and 30 degrees C. Activity is restored by NH2OH. The pH dependence of inactivation reveals that the essential residue modified by DEP has an apparent pKa of 6.5. The difference spectrum of the intact and DEP-treated enzyme shows a maximum between 230 and 240 nm, suggesting formation of carbethoxy derivatives of histidine. The enzyme is also inactivated by N-ethylmaleimide (NEM) and 5,5'-dithiobis-(2-nitrobenzoic acid), both sulfhydryl-modifying agents, with the latter effect reversed by dithiothreitol, which suggests inactivation resulting from modification of cysteine residue(s). Partial inactivation of the enzyme by DEP or NEM results in an apparent decrease in the Vmax without a change in the Km or the extent of CaM stimulation. The rate of inactivation by DEP is greater in the presence than in the absence of Ca/CaM. A substrate analogue, Br-cGMP, and the competitive inhibitor 3-isobutyl-1-methylxanthine partially protect the enzyme against inactivation by DEP or NEM, suggesting that the modification of histidine and cysteine residues occurs at or near the active site. DEP also inactivated porcine brain CaM-PDE.(ABSTRACT TRUNCATED AT 250 WORDS)

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