Mapping the ATP-Binding Site in the Catalytic Subunit of A denosine-3':5'-monophosphate-Dependent Protein Kinase. Spatial Relationship with the ATP Site of the Undissociated Enzyme

Abstract

A set of 24 ATP analogs modified at various positions of the ATP molecule was used for mapping the ATP-binding site in the free catalytic subunit (C) of CAMP-dependent protein kinase (type I). Ki values for these analogs (of which 23 were shown to be competitive with ATP) were measured and compared with Ki values previously obtained for the same set of analogs upon binding to the undissociated form of the enzyme (R2C2). It was found that modifications at the adenine part of ATP bring about a considerable reduction in affinity between C and the resulting analog. The other parts of the ATP molecule play a less important, though definite, role in the binding of this nucleotide to C. By measuring the effect of each given modification in ATP on its binding to C, and comparing the effect of this modification on the binding of the same analog to R2C2, it was possible to obtain 'specificity profiles' for both forms of the kinase. Using such profiles it is shown that the adenine-binding subsite in C may well coincide with the adenine-binding subsite in R2C2. Two plausible models describing the spatial relationship between the ATP sites in C and in R2C2 are proposed.In spite of being catalytically inactive, the undissociated form of CAMP-dependent protein kinase (type I, from rabbit muscle) is known to possess a highaffinity site for ATP (& % 50 nM [l]). It was previously shown [2] that this ATP site does not overlap the cAMP sites on the regulatory subunit of the undissociated enzyme since (a) ATP and cAMP do not compete with each other for binding to the cAMP sites and (b) a variety of substitutions in ATP affect the binding of the resulting analogs to the undissociated enzyme in a way which is different from the way parallel substitutions in cAMP affect the binding of these analogs to the same protein. Furthermore, it was pointed out [2] that the high-affinity ATP site in the undissociated enzyme has some features in common with ATP sites of other phosphotransferase enzymes such as .phosphoglycerokinase and tRNA synthetases [3-71, raising the possibility that this ATP site may overlap the ATP site residing in the catalytic subunit of the enzyme. We wish to report here the K i values for the binding of 24 ATP analogs to the pure catalytic subunit, and compare these values with the Ki values

The ATP Substrate Site of a Cyclic‐Nucleotide‐Independent Protein Kinase from Porcine Liver Nuclei

“…The present work deals with a detailed analysis of the ATP site of this enzyme using 30 ATP analogues. These data have been compared with previous results on the ATP site of the catalytic subunit of CAMP-dependent protein kinase I from rabbit muscle [25,26]. Furthermore, these studies allow specific inhibitors of the nuclear protein kinase to bc determined.…”

“…they can also use CiTP as phosphoryl donor [7,9,10,17,19 -231. This is in contrast to type I enzymes of this group and also to the cyclic-nucleot ide-dependent enzymes which are highly ATP-specific [25].…”

Localization of the High‐Affinity ATP Site in Adenosine‐3′:5′‐monophosphate‐Dependent Protein Kinase Type I

“…Furthermore no site for ATP in the catalytic subunit other than the active site has been found, as revealed by kinetic studies [23,24] and competition experiments with a variety of different analogues [9]. The idea of two different sites in the catalytic subunit for ATP, one site being completely abolished when the other is created, is fairly unlikely.…”

“…As the nature of the high-affinity site for ATP in the holoenzyme and the catalytic subunit are alm'ost identical with respect to their adenine binding domain [9], it seems reasonable to assume that ATP binds to the same domain in both cases. Furthermore no site for ATP in the catalytic subunit other than the active site has been found, as revealed by kinetic studies [23,24] and competition experiments with a variety of different analogues [9].…”

“…Comparing the structural requirements of the ATP site in the holoenzyme and that in the catalytic subunit by mapping experiments with analogues of ATP, we found remarkable similarities of both sites with respect to the adenine moiety. We suggested that ATP binding to the holoenzyme occurs via a binding of the adenine moiety to the catalytic subunit [9].…”