The site and sequence specificity of protein kinases, as well as the role of the secondary structure and surface accessibility of the phosphorylation sites on substrate proteins, was statistically analyzed. The experimental data were collected from the literature and are available on the World Wide Web at http://www.cbs.dtu.dk/databases/PhosphoBase/. The set of data involved 1008 phosphorylatable sites in 406 proteins, which were phosphorylated by 58 protein kinases. It was found that there exists almost absolute Ser/Thr or Tyr specificity, with rare exceptions. The sequence specificity determinants were less strict and were located between positions 34 and +4 relative to the phosphorylation site. Secondary structure and surface accessibility predictions revealed that most of the phosphorylation sites were located on the surface of the target proteins.z 1998 Federation of European Biochemical Societies.
The Michaelis-Menten parameters k,,,, Ks(app) and the second-order rate constants kll = kz/K, of acetylcholinesterase-catalyzed hydrolysis of 25 acetic esters with non-ionic leaving groups have been determined at 25 "C and pH 7.5 in 0.1 5 M KCI. A linear relationship between the substrate noncovalent binding capacity and the leaving group hydrophobicity, and a multiparameter correlation of the acetylation reaction rate constant logarithm with the leaving group inductive effect, hydrophobicity, and steric effect, have been established. The acetyl-enzyme deacetylation rate constant has been calculated. Taken together, a fairly complete understanding of acetylcholinesterase specificity is possible.The data are consistent with a model of the acetylcholinesterase active site, in which the catalytically active groups are located at the bottom of a jaws-like slit with a limited range of hydrophobic walls that provide the sorption of the substrate leaving groups not longer than that in n-butyl acetate.Acetylcholinesterase has been shown to hydrolyse, in addition to its specific substrate acetylcholine, many other acetic esters [l]. This provides a prospect for extensive study of the influence of substrate leaving group structure on the effectiveness of acetylcholinesterase-catalyzed hydrolysis. A conclusion that both the substituent inductive effect and hydrophobicity are important in acetylcholinesterase reactions, can be drawn from the results of previous studies [2-121. In this paper we are concerned with quantitative evaluation of these influences in the reaction series CH3C-(O)OX, where the group X includes hydrocarbon chains and various non-ionic electronegative substituents (see Table 1).Up to now the analysis of enzyme kinetics data by means of correlation equations has been carried out mainly on chymotrypsin-catalyzed reactions [13 -161. In accordance with the three-step reaction scheme(2) (3) the structure-activity relationship for the non-covalent binding step (3) has been given by the equation where K, = k-l/kl, while E is enzyme, S substrate, ES the enzyme-substrate complex, EA the acyl-enzyme, PI and P2 the first and second products. For the acylation reaction (2), as well as for the deacylation (3), the multiparameter correlation equation log k2 = log k2" + @* O* + van + 6 E, ( 5 ) can be applied. The parameters e*, q and 6 in the equations are the intensity factors of the inductive effect, hydrophobicity and steric effect, respectively. The constants cr* and E, have their usual meanings as in physical organic chemistry [17]. The hydrophobicity constants n have been introduced by Hansch [18,19].Under the conditions where the product and substrate inhibition phenomena could be left out of consideration, the reaction sequence (1 -3) would be applicable to the acetylcholinesterase-catalyzed hydrolysis of esters [20]. According to the reaction scheme the meaning of the constants kcat and Ks(app) (K, = K, if k2 < k-l) in the Michaelis-Menten equation depends on the values of the ratio kz/kj as follows : (7)
Neuropeptide galanin and its three G-protein coupled receptors, galanin receptor type 1-galanin receptor type 3 (GalR1-GalR3), are involved in the regulation of numerous physiological and disease processes, and thus represent tremendous potential in neuroscience research and novel drug lead development. One of the areas where galanin is involved is depression. Previous studies have suggested that activation of GalR2 leads to attenuation of depression-like behavior. Unfortunately, lack of in vivo usable subtype specific ligands hinders testing the role of galanin in depression mechanisms. In this article, we utilize an approach of increasing in vivo usability of peptide-based ligands, acting upon CNS. Thus, we have synthesized a series of novel systemically active galanin analogs, with modest preferential binding toward GalR2. We have shown that specific chemical modifications to the galanin backbone increase brain levels upon i.v. injection of the peptides. Several of the new peptides, similar to a common clinically used antidepressant medication imipramine, exerted antidepressant-like effect in forced swim test, a mouse model of depression, at a surprisingly low dose range (< 0.5 mg/kg). We chose one of the peptides, J18, for more thorough study, and showed its efficacy also in another mouse depression model (tail suspension test), and demonstrated that its antidepressantlike effect upon i.v. administration can be blocked by i.c.v. galanin receptor antagonist M35. The effect of the J18 was also abolished in GalR2KO animals. All this suggests that systemically administered peptide analog J18 exerts its biological effect through activation of GalR2 in the brain. The novel galanin analogs represent potential drug leads and a novel pharmaceutical intervention for depression.
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