Knut H. Dahl

The reactivity of affinity labels: A kinetic study of the reaction of alkyl halides with thiolate anions—a model reaction for protein alkylation

Abstract Factors have been investigated which govern the electrophilic reactivity of alkyl halides with thiolate anions in aqueous solution. In the series of alkyl halides studied, some are potential metal-directed affinity labels, while others are frequently used in protein modification. Previous data on the kinetics of this type of alkylation are compared with the present results. The influence of electronic, polar, and steric factors on alkyl halide reactivity is seen. The following order of reactivity for alkyl halides bearing different α substituents was observed: RCH 2 CH(X)COOCH 3 > RCH 2 CH(X)CONH 2 > RCH 2 CH(X)COOH > RCH 2 CH 2 X > RCH 2 CH(X)CH 2 OH. The metal-directed affinity labels are imidazole derivatives, some of which have substituents in their imidazole ring. The effect of the imidazole ring and of ring substitution on reactivity is seen. The nucleophilic reactivity of thiols is highly pH dependent since the thiolate anion (RS − ) is the reactive species, but only minor differences emerged between different free thiolates.

Chemical modification of liver alcohol dehydrogenase with α-bromo-β-(5-imidazolyl) propionic acid

Imidazol is known to form complexes with liver alcohol dehydrogenase, which will stimulate inactivation and inhibition of the enzyme by several reagents [l-5] . Crystallographic studies have confirmed that one imidazol is bound per subunit [5]. The zinc atom at the catalytic centre has three protein ligands, cystelne-46, cysteine-174 and histidine-67 [6] . The fourth ligand is a water molecule/ hydroxyl ion, which can be replaced by imidazole [1,61. Specific modifications of the cysteine residues at the active centre in the horse liver enzyme or the related [7] yeast enzyme, have been performed with different reagents. Among them are iodoacetate [8,9] dibromoacetone [lo] and reactive coenzyme analogues [ 11,121. Specificity depends on the nature of the reagent and is influenced by other factors than just binding at the active site. In this context, cr-bromo/3-(5imidazolyl) propionic acid (BIP) was considered to be of particular interest, since its imidazol group could be expected to bind specifically to the active site zinc atom. This would give a direct correlation between the primary binding site and the secondary chemical modification. BIP has been used previously to modify cysteine residues in papain [ 131. The rate of inactivation of liver alcohol dehydrogenase with BIP is not proportional to the inhibitor concentration, but follows Michaelis-Menten type kinetics [ 141, again supporting specific binding of the reagent. The reaction of BIP with horse liver alcohol dehydrogenase has therefore been studied in the present work, and the modified residue determined. The results show that only one residue, cysteine-46, is selectively modified in the reaction.

Metal-directed affinity labelling. Inactivation and inhibition studies of two zinc alcohol dehydrogenases with twelve imidazole derivatives.

The role of metals in metalloproteins is the object of much research. Metal-directed affinity labels can provide a new way of studying such metals and their near surround~gs, as well as the specific inactivation of met~oenzymes. The general claims for such a reagent are the ability to ligand a metal atom, and the capacity to modify an amino acid residue. A selective modification of amino acid residues in the metalbinding region of the protein may thus be attained. Each subunit of alcohol dehydrogenase from liver contains one structural and one catalytic zinc atom [ 11. The former has four protein ligands while the latter has three to the protein and one free. For the yeast enzyme, ihe exact number of zinc atoms is the subject of discussion [2,3]. The monodentate chelate-binding molecule, imidazole, binds as a free ligand to zinc in the active site of the liver enzyme, and can promote activation or inhibition of the enzyme [4,5]. Imidazole inhibits the yeast enzyme competitively with ethanol (unpublished results). It has been shown that (&Q-2bromo-3(5-imidazolyl) propionic acid (BIP), prior to selective and irreversible alkylation of cysteine-46, binds reversibly at the active site zinc atom of liver alcohol dehydrogenase through the imidazole ring [6,71. The aim of this work has been to survey how a series of metal-d~ected affmity labels react with two metalloenzymes. Twelve imidazole derivatives are surveyed for inhibition and inactivation of two zinc

Stereoselective metal-directed affinity labelling: A model inactivation study with alcohol dehydrogenase from liver

Enzymes, being asymmetric, have chiral active centers, are stereospecific and discriminate between different configurations of substrates, coenzymes and products. Stereoselective alkylation with asymmetric or chiral reagents can likewise result; this has been reported with ribonuclease [1,2], papain [3], yeast alcohol dehydrogenase [4] and yeast giyceraldehydephosphate dehydrogena~ [S]. With these enzymes inactivation showed different alkylation rates with the two enantiomers of haloacids and amides, but absolute discrimination between any of the enantiomers was not attained. The metal-directed affinity label (R,S)-2.bromo-3(5.imidazolyl)propionic acid (BIP), has been shown to label cysteine-46 of liver alcohol dehydrogenase in a two step reaction [6,7]. A reversible complex is first formed as an intermediate by ligand binding to the active-site zinc atom through the imidazole ring. Labelling is no doubt highly dependent upon the conjuration of BIP, but thus far, this racemic label has not been resolved into its enantiomers. However, it has been possible to investigate the stereospecific role of configuration by synthesis of the pure enantiomers of the corresponding chlorocompounds; (R)and (S)-2-chloro3(5Gmidazolyl)propionic acid (CIP), and the methylesters; (R)and @)-methyl 2-chloro-3-(5-imidazolyl)propionate (CIPME). In this work inactivation of liver alcohol dehydrogenase has been attempted with the two enantiomers of CIP and CIPME. The reaction was totally stereoselective or stereospecific. No reaction

Metal-directed affinity labeling of zinc(II), cobalt(II) and cadmium(II) horse liver alcohol dehydrogenases

The active site metal in horse liver alcohol dehydrogenase has been studied by metal-directed affinity labeling of the native zinc(II) enzyme and that substituted with cobalt(II) or cadmium(II). Reversible binding of bromoimidazolyl propionic acid to the cobalt enzyme blueshifts the visible absorption band originating from the catalytic cobalt atom at 655 to 630 nm. Binding of imidazole to the cobalt(II) enzyme redshifts the 655 nm band to 667 nm. Addition to bromoimidazolyl propionic acid blueshifts this 667 nm band back to 630 nm. This proves direct binding of the label to the active site metal in competition with imidazole. The affinity of the label for the reversible binding site in the three enzymes follows the order Zn greater than Cd greater than Co. After reversible complex formation, bromoimidazolyl propionic acid alkylates cysteine-46, one of the protein ligands to the active site metal. The nucleophilic reactivity of this metal-mercaptide bond in each reversible complex follows the order Co greater than Zn greater than Cd.

Reactivation of apo horse liver alcohol dehydrogenase with the monovalent metal ion Ag(I)

Each subunit of the liver alcohol dehydrogenase dimer contains one catalytic and one structural Zn(II) atom. Enzyme with the catalytic metal atoms selectively removed is inactive but can be partly reactivated in the presence of Ag(I) ions. Reactivation results from Ag(1) ions entering the empty metal-binding site in the catalytic center. The specific activity of this silver enzyme reached 24% of the native enzyme. Atomic absorption analysis gave equal amounts of Ag(I) and Zn(II), corresponding to one mole of each metal per monomer. Metal-directed affinity labelling using bromo-imidazolyl propionate showed that the properties of the silver-reactivated enzyme were distinct from those of the native enzyme.

Metal-Directed Affinity Labelling and Promoted Alkylation of a Thiol Liganded to the Catalytic Metal Ion in Liver Alcohol Dehydrogenase

Cystine-46, which is a protein ligand to the catalytic metal atom in liver alcohol dehydrogenase, has been selectively alkylated with several alkyl-ha1oacids. When imidazole replaces H2O as a free ligand to this metal atom, the alkylation rate increases, A thermodynamic transitionstate analysis of enzyme alkylation showed that the effect of imidazole was due to a decreased enthalpy barrier. Imidazole also promoted or stimulated the alkylation with iodoacetate of the model-compound zinc-mercaptoethano 1. Alkylation is promoted by imidazole donating electrons to the catalytic metal atom which distributes the increased electron density to the other ligands. Metal-directed affinity labelling with bromo-imidazoly1 propionate has been carried out on liver alcohol dehydrogenase substituted with several metal atoms in the active site. Since the stability of the reversible complex and the nucleophilic reactivity of Cys-46 depends on metal type, meta1-directed affinity labelling delineates different active site properties of different metalloenzyme species.