Butyl isocyanate, an active-site-specific reagent for yeast alcohol dehydrogenase

Twu, Jer-Shung; Wold, Finn

doi:10.1021/bi00727a003

Cited by 55 publications

(21 citation statements)

References 15 publications

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“…The shift involving residues 119-139 (Fig.1) in the horse enzyme is required by considerations of both homology and function. Thus, by inclusion of this shift, the succeeding segment in the comparison is one of those showing maximum similarity, and a functionally important cysteine residue at the active site of either enzyme (cysteine-174 in the horse and cysteine-153 in the yeast protein [11,36,39,40]) is at equivalent positions ( Fig. 1).…”

Section: Alignmentmentioning

confidence: 99%

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Differences between Alcohol Dehydeogenases

Jörnvall

1977

European Journal of Biochemistry

110

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Comparisons of the primary structures of yeast and horse liver alcohol dehydrogenases reveal that the enzymes are homologous but distantly related. The overall positional identity is 25 between common regions, and several deletions/insertions occur in either enzyme, the longest apparently corresponding to 21 residues, showing that the different subunit sizes are largely explained by internal differences.Variabilities in the structural similarities can be coupled with functional requirements but not directly with whole domains in the previously known tertiary structure of the horse protein. The two most similar regions of the enzymes affect active-site segments and the two most dissimilar regions seem to affect a loop structure without known function, and a segment participating in subunit interactions. The dissimilarities may probably be correlated with changes in zinc-binding properties and quaternary structures.The extra region corresponding to the large internal chain-length difference shows an apparent coincidence in sequence to a following segment of the horse enzyme, and additional elements of internal coincidences, or superficial similarities with other dehydrogenases, are noticed. These characteristics are not fully distinguishable from chance distributions but in view of the extensive species variations in alcohol dehydrogenases some evolutionary considerations may not be excluded, in which case a model relating all regions of these and associated enzymes to a common ancestor is shown to be compatible with all known observations.

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Section: Alignmentmentioning

confidence: 99%

“…1). Finally, particular structures in the ancestral unit or an early descendant form, could explain the positions of cysteine residues, some of which are important [23,24,39,40] although functionally completely different [4], and the mutually similar cysteine-tryptophan distances.…”

Section: Domain Relationshipsmentioning

confidence: 99%

Differences between Alcohol Dehydeogenases

Jörnvall

1977

European Journal of Biochemistry

110

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“…The pK values of these groups are in the pH range 6.5 -9.0, which is consistent with imidazole and thiol groups being involved in catalysis. Thus the essential histidine residue described in this paper, the essential thiol group [1,9,16] and the butylisocyanate-sensitive thiol group [20,33], all of which are required for the formation of ternary, but not binary complexes, may all be at least partly responsible for the variation with pH of the rate of the catalytic step.…”

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confidence: 99%

The Role of an Essential Histidine Residue of Yeast Alcohol Dehydrogenase

Dickenson

Dickinson

1975

European Journal of Biochemistry

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1. Inactivation of yeast alcohol dehydrogenase by diethyl pyrocarbonate indicates that one histidine residue per enzyme subunit is necessary for enzymic activity. The inactivated enzyme regains its activity over a period of days. . pyrazole, which are characteristic of native enzyme. 3.The rate constant for the reaction of enzyme with diethyl pyrocarbonate has been determined over the pH range 5.5-9. The histidine residue involved has approximately the same pK, as free histidine, but is 10-fold more reactive than free histidine.Alcohol dehydrogenases from yeast and horse liver each possess a histidine residue, five residues removed from a cysteine residue which, on alkylation with iodoacetate or iodoacetamide, results in the total loss of enzymic activity [l]. It is possible that a histidine residue may be important in catalysis by both of these alcohol dehydrogenases. Accordingly, we have studied the reaction of yeast alcohol dehydrogenase with diethyl pyrocarbonate under conditions where this reagent reacts with histidine residues [2]. Other pyridine-nucleotide-dependent dehydrogenases are known to be inactivated by diethyl pyrocarbonate, e.g. pig heart lactate dehydrogenase [3] and ox liver glutamate dehydrogenase [4]. This paper presents studies on the inactivation of yeast alcohol dehydrogenase by diethyl pyrocarbonate, and on some of the properties of the modified enzyme. The interaction of diethyl pyrocarbonate with horse liver alcohol dehydrogenase has also been examined. A preliminary report of some of the work has been published [5]. MATERIALS AND METHODS MaterialsAll solutions were prepared using glass-distilled water. The sodium phosphate and sodium pyrophos-

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“…This is an important consideration in the case of the imidazole nitrogen reaction since although it is a good nucleophile at physiological pH, the reaction of secondary amines is slow relative to the rate of isocyanate hydrolysis. Of particular interest is the reaction with the sulfhydryl group that can occur at physiological pH, whose product is stable under acidic conditions, but which is fully reversible under slightly alkaline pH conditions (8). It is worth noting that the reversal of this reaction proceeds through formation of the isocyanate (4).…”

mentioning

confidence: 99%

“…Subsequent to this work, in vitro titrations of other enzymes were performed; noteworthy among these were papain and alcohol dehydrogenase (8), which were both inhibited by butyl isocyanate. The major differences between these studies and those previously cited for the serine esterases are that the inhibition of both of these enzymes results from modification of their active site sulfhydryl group and that the inhibition by the isocyanates was reversible under slightly alkaline pH (pH : 7.0).…”

mentioning

confidence: 99%

Biochemistry of protein-isocyanate interactions: a comparison of the effects of aryl vs. alkyl isocyanates.

Brown¹,

Green²,

Cedel³

et al. 1987

Environ Health Perspect

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In addition to their use in the polyurethane and pesticide industries, isocyanates have proven to be useful probes for the exploration of protein structure. This paper focuses on three aspects of isocyanates: their broad reactivity, their reversible interaction with cholinesterases, and the relative hydrolysis rates of alkyl and aryl isocyanates. The broad reactivity of isocyanates as well as the demonstrated affinity labeling of serine and sulfhydryl esterases are discussed. Extension of the affinity labeling studies to include the analysis of the inhibition of cholinesterases by methyl isocyanate shows that methyl isocyanate is not an effective inhibitor of any of the cholinesterases. The inhibition of cholinesterases by alkyl isocyanates shows a pattern of decreased specificity with decreased alkyl chain length. The inhibition of cholinesterases by isocyanates is shown to be reversible, with a maximum rate of reversal seen at physiological pH. This reversal is characteristic of the reaction of an isocyanate with a sulfhydryl group. Finally, the affinity labeling of proteins must compete successfully with the hydrolysis of isocyanates in aqueous solution. The hydrolysis of alkyl isocyanates is shown to be significantly slower than that of the aryl isocyanates.The application of alkyl and aryl isocyanates to the pesticide and polyurethane industries is of major commercial interest worldwide. In addition, the isocyanates have proven to be valuable tools to protein chemists making correlations between protein structure and function. It is the information from these latter studies that may prove valuable to our understanding of how isocyanates affect human and animal populations. As a reagent used for the study of protein structure and function, isocyanates have two major characteristics that make them attractive. First, they are highly reactive with a variety of functional groups found on biological macromolecules, and second, they have a finite lifetime that enables them to react with selected functional groups but not to exist long enough to cause significant, nonspecific modification of the macromolecule under study. It is the purpose of this paper to explore both of these advantages of the isocyanates as protein modification reagents, to summarize information already in the literature, and to present new data relevant to each point. Of particular interest to this symposium will be the comparison of the biochemical results for the aryl isocyanates and the alkyl isocyanates. In the former case, the emphasis will be on toluene diisocyanate (TDI)

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Butyl isocyanate, an active-site-specific reagent for yeast alcohol dehydrogenase

Cited by 55 publications

References 15 publications

Differences between Alcohol Dehydeogenases

Differences between Alcohol Dehydeogenases

The Role of an Essential Histidine Residue of Yeast Alcohol Dehydrogenase

Biochemistry of protein-isocyanate interactions: a comparison of the effects of aryl vs. alkyl isocyanates.

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