Aqueous and nonaqueous chemical approaches to elucidating structure and function in proteins.

Abstract

Three chemical approaches were used to study proteins: Chemical modification with iodomethane: A novel strategy was developed in which proteins were simultaneously reacted with $\rm\lbrack \sp C\rbrack $ and $\rm\lbrack \sp C\rbrack $iodomethane to permit the identification of reactive groups by $\rm\lbrack \sp C\rbrack $NMR spectroscopy and the isolation of $\rm\lbrack \sp C\rbrack $labeled peptides containing individual methylated functional groups by autoradiography. Reaction with iodomethane N,N,N-trimethylated $\rm N\varepsilon$-lysyl and N-terminal $\rm N\alpha$-amino groups, $\rm N\sp1,N\sp3$-dimethylated imidazole functions of histidyl residues, O-methylated phenolic hydroxyl functions of tyrosyl residues and S-methylated sulphide functions of methionyl residues. Nonaqueous chemical modification techniques: Chemical modification of lyophilized proteins in nonaqueous environments was carried out by either dispersing protein in octane and reacting with dissolved reagent, or reacting protein in vacuo with a volatile reagent. The reaction of iodomethane with protein functional groups in nonaqueous environments and the derivatives formed paralleled those of aqueous reactions and were quantified by solution and solid state $\rm \lbrack \sp C\rbrack $NMR techniques. Reacting ethoxyformic anhydride or acetic anhydride with protein functional groups afforded acyl derivatives of $\rm N\alpha$ and $\rm N\varepsilon$-amino groups and mixed anhydrides of side-chain carboxyl groups. These mixed anhydrides were stable in the absence of water and enabled the coupling of protein to various nucleophiles. The extent of derivatization of functional groups of lyophilized protein was directly related to the $\rm pK\sb{a}$ of the reactive group, pH of the solution from which the protein was lyophilized, and extent of surface exposure of functional groups under native conditions. Enzyme kinetics: Five synthetic substrates containing different amino acid residues at the $\rm P\sb3$ position (acetyl-X-Arg-Arg-AMC, where X is Gly, Glu, Arg, Val and Tyr and where AMC represents 7-amido-4-methylcoumarin) were used to investigate the $\rm S\sb3$ subsite specificity of cathepsin B. At pH 6.0, the specificity constant, $k\sb{cat}/K\sb{\rm m},$ for tripeptide substrate hydrolysis was observed to increase in the order $\rm Glu Gly Arg Val Tyr.$ Molecular modeling studies of substrates containing a $\rm P\sb3$ Glu, Arg or Tyr covalently bound as the tetrahedral intermediate to cathepsin B suggest that enzyme specificity for a $\rm P\sb3$ Tyr group is due to a favourable aromatic-aromatic interaction with $\rm Tyr\sp{75}$ on the enzyme as well as a possible hydrogen bond between the $\rm P\sb3$ Tyr hydroxyl and the side-chain carboxyl of $\rm Asp\sp{69}.$ (Abstract shortened by UMI.)