We have addressed the question of whether protein kinase substrate efficacy is a reliable barometer for successful inhibitor design by assessing the dependence of kcat and kcat/Km for eight separate alcohol-bearing residues on solvent viscosity. We have found that the Km for three structurally distinct primary alcohol-containing peptides overestimates the affinity that these species exhibit for the cAMP-dependent protein kinase. In all three cases, the rate-determining step is product release, and substrate binding is best described as rapid equilibrium. In contrast, peptides containing the following phosphorylatable residues all provide Km values that are accurate assessments of substrate affinity for the protein kinase: a secondary alcohol, a simple phenol, and a primary alcohol with a relatively long side chain. In the latter three instances, the rate-determining step is phosphoryl transfer. Finally, two aromatic alcohol-containing residues that possess lipophilic side chains exhibit Michaelis constants that underestimate enzyme affinity. These results demonstrate that while it may be tempting to employ structural elements from the most efficient substrates (e.g. primary alcohols) for inhibitor design, less effective substrates may serve as a more accurate assessment of inhibitory success.
This work was supported by Research Grant GM45989 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
