Conformationally Restricted Creatine Analogs and Substrate Specificity of Rabbit Muscle Creatine Kinase

Robert F. Dietrich, Robert B. Miller, George L. Kenyon, Thomas S. Leyh, George H. Reed

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

Several conformationally restricted analogues of creatine have been both synthesized and examined as potential substrates or inhibitors of rabbit muscle creatine kinase (EC 2.7.3.2). When an asymmetric center was included in a creatine analogue in the position α to the carboxyl group, the enzyme had a pronounced preference for the R enantiomer. Thus, whereas (R)-N-amidinoazetidine-2-carboxylic acid (7) has been shown to be a good substrate (Ks = 72 mM, Km = 39 mM, and Vmax = 29% relative to that of creatine) for creatine kinase, the corresponding S enantiomer 6 showed only barely detectable reactivity (Fmax(rel) ≪ 1%). When the corresponding ring-opened analogue, N-methyl-N-amidino-alanine, was examined as a substrate, creatine kinase again showed a strong preference for the R enantiomer 9 [Ks = 94 mM, Km = 82 mM, Vmax(rel) ≃ 10%]. The R enantiomer was approximately 7 times more reactive than its S enantiomer 8 when they were both examined as substrates at 40 mM in the presence of 4 mM ATP. On the other hand, the conformationally restricted creatine analogues N-[2-(4,5-dihydroimidazolyl)]sarcosine (10), 2-iminoimidazolidine-4-carboxylic acid (11), and 2-imino-3-methylimidazolidine-4-carboxylic acid (12) did not show detectable activity either as substrates or as inhibitors. Binding studies of the (R)-N-amidino-azetidine-2-carboxylic acid (7) and its corresponding S enantiomer 6 in dead-end complexes with MnADP and in an-ion-stabilized dead-end complexes revealed that the affinity of the enzyme for 7 was between one and two orders of magnitude greater than for its S enantiomer 6. Unlike several other analogues of creatine, analogue 7 gave EPR spectra in the anion-stabilized complexes with MnADP that were virtually identical with those for creatine.

Original languageEnglish (US)
Pages (from-to)3180-3186
Number of pages7
JournalBiochemistry
Volume19
Issue number14
DOIs
Publication statusPublished - Jan 1 1980

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ASJC Scopus subject areas

  • Biochemistry

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