Mapping the ATP-binding site in the catalytic subunit of adenosine-3':5'-monophosphate-dependent protein kinase. Spatial relationship with the ATP site of the undissociated enzyme.

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 R2C2 are proposed.

Mechanism of activation of the protein kinase I from rabbit skeletal muscle. The high-affinity ATP site of the holoenzyme.

The high-affinity binding site for ATP of the holoenzyme of cAMP-dependent protein kinase (type I) from rabbit skeletal muscle has been investigated. Binding affinity of a series of ATP derivatives substituted at different sites in the molecule was determined by competition with tritiated ATP. The results were compared with data available from cAMP derivatives with the same substituents, in order to analyse the electronic and steric features of these two sites on the protein kinase. The comparison revealed significant differences of the effect of substituents towards the two sites. In particular the N6-derivatives of ATP and substituents affecting the gamma-phosphate indicate that the high-affinity ATP site of the protein kinase has similar properties as those found for phosphotransferase sites. The present results are consistent with the supposition that the high-affinity site for ATP on the holoenzyme is congruent with the phosphotransferase site of the catalytic subunit. Upon combination of catalytic and regulatory subunits this site would be transformed into a high-affinity site for ATP with simultaneous blocking of the phosphotransferase activity.

Influences of amino acid, ATP, pyrophosphate and tRNA on binding of aminoalkyl adenylates to isoleucyl-tRNA synthetase from Escherichia coli MRE 600.

Aminoalcohol-AMP esters, structurally related to the assumed intermediates of the amino acid activation reaction, behave as competitive inhibitors both with respect to the amino acid and ATP, when tested in the ATP-(32P) PPi-exchange or the tRNA-charging reaction. However, closer investigation of the binding of norvalinyl adenylate to isoleucyl-tRNA synthetase from Escherichia coli MRE 600 by an equilibrium method shows that only the amino acid is a true competitor, while ATP cannot displace the ester from binding. Pyrophosphate enhances the stability of the ester-enzyme complex whereas tRNA is without detectable influence.

On the binding of aminoalkyl adenylates to isoleucyl-tRNA synthetase from Escherichia coli MRE 600.

The binding of nine aminoalkyl adenylates to isoleucyl-tRNA synthetase from Escherichia coli MRE 600 was measured and compared with the binding of the cognate amino acids. It was found that they bind rather tightly to the enzyme, the Kd's ranging from 3.1.10(-4) M with glycinol-AMP ester to 3.7.10(-9) M with L-isoleucinol-AMP ester. The binding is not affected by magnesium. It is shown that the free energies of binding of the esters can be calculated adding a constant contribution of the AMP-moiety of about - 4.1 (- 17) kcal/mole (kJ/mole) to the free energies of binding of the cognate amino acids, which we have reported earlier (19, 25, 26).

ATP-analogues as substrates for the leucyl-tRNA synthetase from Escherichia coli MRE 600.

No analogous nucleoside triphosphate was found which acts as well as ATP in binding to and supporting catalysis of leucyl-tRNA synthetase from Escherichia coli MRE 600. However, there are numerous nucleotides which are able to replace ATP, but with lower efficiency. The 6-amino group of the adenine ring and the 2'-hydroxyl group of the ribose ring are essential for binding and catalytic activity. Alterations in the triphosphate moiety of the molecule can cause drastic changes in Km and/or Vmax, whereas alterations of the imidazole ring and substitutions at the 8-position of the adenine ring cause only minor losses of catalytic activity.