A kinetic study of cyclic adenosine 3':5'-monophosphate binding and mode of activation of protein kinase from Drosophila melanogaster embryos

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Abstract
Cyclic[c]AMP-dependent protein kinase and its regulatory subunit were isolated from D. melanogaster embryos. The profiles of cAMP binding by these proteins were significantly different. In order to explain such a difference and to find the mode of enzyme activation by cAMP, a kinetic study of cAMP binding was carried out. The association rate constant k1 and dissociation rate constant k-1 in the cAMP-regulatory subunit interaction at 0.degree. C were estimated to be 2.3 .times. 106 M-1 s-1 and 1.1 .times. 10-3 s-1, respectively. The 3 possible modes of enzyme activation by cAMP were mathematically considered and could be described by a unique formula: r = APt + BQt (A + B = 1) in which the parameters A, B, P and Q were equivalent to rate constants in the sense that the rate constants were simply expressed by these parameters. The values of the parameters and subsequently, values of rate constants involved in the possible mechanisms were evaluated using a curve-fitting technique and compared with experimental observation. The only mechanism which fitted the experimental observations was .**GRAPHIC**. where R, C and L represented the regulatory and catalytic subunits and cAMP as a ligand. Results indicated that in the presence of cAMP the active enzyme (C) was released from a ternary intermediate which was the primary product of the cAMP-holoenzyme interaction. The estimated values of the rate constants were: k3 = 3.5 .times. 106 M-1 s-1; k-3 = 7.3 .times. 10-1 s-1; and k4 = 3.8 .times. 10-2 s. The reaction LRC .fwdarw. RL + C seemed relatively slow and limited the rate of the overall reaction. By comparing k-3 and k4, it was apparent that a large part of newly formed ternary intermediate reverted to the holoenzyme.