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Biochemistry. 1994 Nov 1;33(43):12807-20.

Mechanism of 2-chloro-(epsilon-amino-Lys75)-[6-[4-(N,N- diethylamino)phenyl]-1,3,5-triazin-4-yl]calmodulin interactions with smooth muscle myosin light chain kinase and derived peptides.

Author information

1
National Institute for Medical Research, Mill Hill, London, United Kingdom.

Abstract

The mechanism of the interactions of 2-chloro-(epsilon-amino-Lys75)-[6-[4-(N,N-diethylamino)phenyl]- 1,3,5-triazin-4-yl]calmodulin (TA-calmodulin) with smooth muscle myosin light-chain kinase (MLCK) and two 17-residue peptides, Ac-R-R-K-W-Q-K-T-G-H-A-V-R-A-I-G-R-L-CONH2 (Trp peptide) and Tyr peptide, in which W is replaced by Y, were studied by measurements of equilibrium and transient fluorescence changes in the nanomolar range. Most reactions were carried out in 100 microM CaCl2 at ionic strength 0.15 M, pH 7.0, and 21 degrees C. In each case association of MLCK or peptide to TA-calmodulin could be described by a two-step process, a bimolecular step and an isomerization. In the case of the interaction between TA-calmodulin and Tyr peptide it was shown that the isomerization involved the binary complex of TA-calmodulin and Tyr peptide as opposed to an isomerization of either TA-calmodulin or Tyr peptide in isolation. These distinctions depended in part on development for transient kinetic experiments of a general theory to quantify relative phase amplitudes in two-step mechanisms. The kinetics for all three association reactions were then interpreted in terms of a bimolecular association (rate constants k+1 and k-1) followed by an isomerization of the binary complex (rate constants k+2 and k-2). For the interaction of TA-calmodulin and Tyr peptide, values of the rate constants are k+1, 8.8 x 10(8) M-1 s-1; k-1, 5.7 s-1; k+2, 0.38 s-1; and k-2, 0.65 s-1. The fluorescence intensities (lambda ex 365 nm, lambda ex 365 nm, lambda em > 400 nm) of TA-calmodulin, the initial binary complex of TA-calmodulin and Tyr peptide, and the isomerized binary complex are in the ratio 1:2.8:1.3. Analogous mechanisms were found for TA-calmodulin binding to Trp peptide and to MLCK, but values for the rate constants and relative fluorescence intensities of the binary complexes were generally not so completely defined. Values for the Trp peptide and MLCK, respectively, are k+1, 8.8 x 10(8) M-1 s-1 and 1.1 x 10(8) M-1 s-1; (k+2 + k-2), 0.97 s-1 and 1.3 s-1; and k-1k-2/(k+2 + k-2), 0.0079 s-1 and 0.025-0.056 s-1. Equilibrium dissociation constants (Kd) for interactions of TA-calmodulin and targets determined from these data are Tyr peptide, 4.1 nM; Trp peptide, 0.011 nM; and MLCK, 0.23-0.51 nM.(ABSTRACT TRUNCATED AT 400 WORDS).

PMID:
7947686
DOI:
10.1021/bi00209a012
[Indexed for MEDLINE]

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