Structure of the unliganded cAMP-dependent protein kinase catalytic subunit from Saccharomyces cerevisiae.

The structure of TPK1delta, a truncated variant of the cAMP-dependent protein kinase catalytic subunit from Saccharomyces cerevisiae, was determined in an unliganded state at 2.8 A resolution and refined to a crystallographic R-factor of 19.4%. Comparison of this structure to that of its fully liganded mammalian homolog revealed a highly conserved protein fold comprised of two globular lobes. Within each lobe, root mean square deviations in Calpha positions averaged approximately equals 0.9 A. In addition, a phosphothreonine residue was found in the C-terminal domain of each enzyme. Further comparison of the two structures suggests that a trio of conformational changes accompanies ligand-binding. The first consists of a 14.7 degrees rigid-body rotation of one lobe relative to the other and results in closure of the active site cleft. The second affects only the glycine-rich nucleotide binding loop, which moves approximately equals 3 A to further close the active site and traps the nucleotide substrate. The third is localized to a C-terminal segment that makes direct contact with ligands and the ligand-binding cleft. In addition to resolving the conformation of unliganded enzyme, the model shows that the salient features of the cAMP-dependent protein kinase are conserved over long evolutionary distances.

Crystal structure of a conformation-selective casein kinase-1 inhibitor.

Members of the casein kinase-1 family of protein kinases play an essential role in cell regulation and disease pathogenesis. Unlike most protein kinases, they appear to function as constitutively active enzymes. As a result, selective pharmacological inhibitors can play an important role in dissection of casein kinase-1-dependent processes. To address this need, new small molecule inhibitors of casein kinase-1 acting through ATP-competitive and ATP-noncompetitive mechanisms were isolated on the basis of in vitro screening. Here we report the crystal structure of 3-[(2,4,6-trimethoxyphenyl) methylidenyl]-indolin-2-one (IC261), an ATP-competitive inhibitor with differential activity among casein kinase-1 isoforms, in complex with the catalytic domain of fission yeast casein kinase-1 refined to a crystallographic R-factor of 22.4% at 2.8 A resolution. The structure reveals that IC261 stabilizes casein kinase-1 in a conformation midway between nucleotide substrate liganded and nonliganded conformations. We propose that adoption of this conformation by casein kinase-1 family members stabilizes a delocalized network of side chain interactions and results in a decreased dissociation rate of inhibitor.

Investigation of ionizable residues critical for sequence-specific enzymatic DNA modification: protein modification and steady-state and pre-steady-state kinetic pH analyses of EcoRI DNA methyltransferase.

Steady-and pre-steady-state pH kinetic analyses are widely used methods to investigate important ionizable groups in enzyme-catalyzed reactions. The first such analysis to identify ionizable residues critical for sequence-specific modification of DNA is presented. EcoRI DNA methyltransferase uses S-adenosyl-L-methionine (AdoMet) to catalyze the N6 methylation of the second adenine in the double-stranded DNA sequence GAATTC. The kinetic mechanism was previously shown to be steady-state-ordered bi bi in which AdoMet binds first followed by DNA addition [Reich, N. O., & Mashhoon, N. (1991) Biochemistry 30, 2933-2939]. Steady-state parameters are strongly dependent on pH and implicate at least four residues with pKa values between 8.2 and 8.9 in the free enzyme and AdoMet-Bound enzyme and one residue with an apparent pKa of 6.0. The data obtained are consistent with the enzyme binding the form of AdoMet in which the alpha amino group is protonated. Two protein residues with an apparent pKa between 8.9 and 9.2 were implicated within the central complex (enzyme-DNA-AdoMet). The general insensitivity of all steady-state parameters to pH changes between pH 6.0 and 8.0 suggests that no critical protein residues undergo ionization-state changes in this range. The lack of significant pH-dependent changes in protein fluorescence and DNA thermal stability suggests minimal structural changes in either macromolecule. In support of the steady-state results single-turnover experiments reveal minimal pH dependence of the methylation rate constant between pH 5.53 and 8.6. Thus, no amino acids critical for catalysis undergo ionization-state changes in this range.(ABSTRACT TRUNCATED AT 250 WORDS)

Presteady state kinetics of an S-adenosylmethionine-dependent enzyme. Evidence for a unique binding orientation requirement for EcoRI DNA methyltransferase.

We present the first presteady state kinetic analysis of an S-adenosylmethionine-dependent enzyme. The target enzyme is the bacterial EcoRI DNA methyl-transferase, which transfers the methyl group to the second adenine in the DNA sequence GAATTC. The rate constant for conversion of the central complex (enzyme-DNA-S-adenosylmethionine) to products (enzyme-methylated DNA-S-adenosylhomocysteine) (41 +/- 7 s-1) is over 300-fold faster than kcat, consistent with our demonstration that steps after methyl transfer are rate-limiting (Reich, N. O., and Mashhoon, N. (1991) Biochemistry 30, 2933-2939). Methyl transfer at the N6 amino moiety of adenine on each strand requires a single binding orientation.

Kinetic mechanism of the EcoRI DNA methyltransferase.

We present a kinetic analysis of the EcoRI DNA N6-adenosine methyltransferase (Mtase). The enzyme catalyzes the S-adenosylmethionine (AdoMet)-dependent methylation of a short, synthetic 14 base pair DNA substrate and plasmid pBR322 DNA substrate with kcat/Km values of 0.51 X 10(8) and 4.1 X 10(8) s-1 M-1, respectively. The Mtase is thus one of the most efficient biocatalysts known. Our data are consistent with an ordered bi-bi steady-state mechanism in which AdoMet binds first, followed by DNA addition. One of the reaction products, S-adenosylhomocysteine (AdoHcy), is an uncompetitive inhibitor with respect to DNA and a competitive inhibitor with respect to AdoMet. Thus, initial DNA binding followed by AdoHcy binding leads to formation of a ternary dead-end complex (Mtase-DNA-AdoHcy). We suggest that the product inhibition patterns and apparent order of substrate binding can be reconciled by a mechanism in which the Mtase binds AdoMet and noncanonical DNA randomly but that recognition of the canonical site requires AdoMet to be bound. Pre-steady-state and isotope partition analyses starting with the binary Mtase-AdoMet complex confirm its catalytic competence. Moreover, the methyl transfer step is at least 10 times faster than catalytic turnover.

Inhibition of EcoRI DNA methylase with cofactor analogs.

Four analogs of the natural cofactor S-adenosylmethionine (AdoMet) were tested for their ability to bind and inhibit the prokaryotic enzyme, EcoRI adenine DNA methylase. The EcoRI methylase transfers the methyl group from AdoMet to the second adenine in the double-stranded DNA sequence 5'GAATTC3'. Dissociation constants (KD) of the binary methylase-analog complexes obtained in the absence of DNA with S-adenosylhomocysteine (AdoHcy), sinefungin, N-methyl-AdoMet, and N-ethylAdoMet are 225, 43, greater than 1000, and greater than 1000 microM, respectively. In the presence of a DNA substrate, all four analogs show simple competitive inhibition with respect to AdoMet. The product of the enzymic reaction, AdoHcy, is a poor inhibitor of the enzyme (KI(AdoHcy) = 9 microM; KM(AdoMet) = 0.60 microM). Two synthetic analogs, N-methyl-AdoMet and N-ethyl-AdoMet, were also shown to be poor inhibitors with KI values of 50 and greater than 1000 microM, respectively. In contrast, the naturally occurring analog sinefungin was shown to be a highly potent inhibitor (KI = 10 nM). Gel retardation assays confirm that the methylase-DNA-sinefungin complex is sequence-specific. The ternary complex is the first sequence-specific complex detected for any DNA methylase. Potential applications to structural studies of methylase-DNA interactions are discussed.