Stereoselective metal‐directed affinity labelling

Dahl, Knut Helkås; McKinley-McKee, John S.; Beyerman, H. C.; Noordam, A.

doi:10.1016/0014-5793(79)80980-1

Cited by 10 publications

(7 citation statements)

References 11 publications

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“…However, with the other enantiomer R-CIP, no reaction was observed and the curve was indistinguishable from the control with no inactivator added. This shows, in agreement with previous results (Dahl et al, 1979), that inactivation with CIP is enantioselective, since inactivation is observed only with the S-enantiomer.…”

Section: Enzyme Inactivation With Cipsupporting

confidence: 93%

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Enantioselective affinity labelling of horse liver alcohol dehydrogenase. Correlation of inactivation kinetics with the three-dimensional structure of the enzyme

Dahl¹,

Eklund²,

McKinley-McKee³

1983

Biochemical Journal

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Kinetic data for the inactivation of horse liver alcohol dehydrogenase with S-2-chloro-3-(imidazol-5-yl)propionate at pH8.2 were correlated with the three-dimensional structure of the enzyme. The R-2-chloro-3-(imidazol-5-yl)propionate enantiomer did not inactivate the enzyme, and the reaction is thus enantioselective. Inactivation follows an affinity-labelling mechanism where a reversible complex is formed before the irreversible alkylation and inactivation of the enzyme. A reversible complex is also formed with the non-inactivating enantiomer, and this shows that the selectivity occurs at the irreversible step. By using a computer-controlled display system, models of the two enantiomers of 2-chloro- and 2-bromo-3-(imidazol-5-yl)propionate were built into a model of the enzyme so that the imidazole moiety was liganded to the active-site metal, while the carboxylate group interacted with the general anion-binding site. The conformation of the imidazole derivatives and their orientation in the active site were adjusted to minimize unfavourable steric interactions. It was clear that alkylation of cysteine-46 could proceed with the S-enantiomer bound in this way, but not with the R-enantiomer. Model building thus agrees with the inactivation kinetics and indicates the structural origin of the enantioselectivity.

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Section: Enzyme Inactivation With Cipsupporting

confidence: 93%

“…The reaction turned out to be totally enantioselective, since S-CIP inactivated the enzyme, whereas with R-CIP no reaction was observed (Dahl et al, 1979).…”

Section: Lb)mentioning

confidence: 97%

Enantioselective affinity labelling of horse liver alcohol dehydrogenase. Correlation of inactivation kinetics with the three-dimensional structure of the enzyme

Dahl¹,

Eklund²,

McKinley-McKee³

1983

Biochemical Journal

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“…As pointed out previously [3], this compound binds reversibly to the active site of the enzyme not only by charge interaction with the general anion-binding site, but also with the imidazole ring liganding to the active-site metal. Bound in this way, the molecule is anchored at both ends and no movement of the arm, as with the other haloacids, is allowed.…”

Section: Enzyme-catalyzed a Lkylut Ionmentioning

confidence: 55%

“…4-6. The reversible complexes are formed by carboxylate binding to the general anionbinding site, and for bromo-imidazolyl propionate also by ligand-binding to the active-site metal [3]. For all the haloacids, the residue alkylated is Cys-46, even if this has not been chemically proved with bromoacetate, 2-bromopropionate, 2-bromobutyrate and 3-bromopropionate.…”

Section: Mechanism Of Enzyme Inactivationmentioning

confidence: 99%

Enzymatic Catalysis in the Affinity Labelling of Liver Alcohol Dehydrogenase with Haloacids

Dahl

McKinley-McKee

1981

Eur J Biochem

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In this work, the inactivation at pH 7.0 of liver alcohol dehydrogenase by iodoacetamide and a series of six haloacids has been studied, and the kinetic constants determined. Enzyme inactivation was compared with the model alkylation of a metal-thiol and a thiolate anion free in solution. The following conclusions resulted.1. Inactivation of liver alcohol dehydrogenase by iodoacetamide is a direct thiol alkylation, while inactivation by selective alkylation of Cys-46 by the haloacids is facilitated by reversible complex formation.2. Inactivation half-time for the haloacids ranged over 4-190 min, a difference mainly caused by dissimilar chemical reactivities rather than diverse fitting in the active site.3. The thiol of Cys-46 is alkylated as a zinc-thiol complex. It is, as such, not especially reactive; indeed it has a nucleophilic reactivity similar to that observed with the model compound free in solution.4. Affinity labelling of liver alcohol dehydrogenase by haloacids compared with alkylation of the similar group free in solution illustrates enzymatic catalysis by reversible complex formation. With the present series of 'substrates' a rate enhancement of up to 58000 is seen.The a-haloacids iodoacetate, bromoacetate, bromoimidazolyl propionate and chloro-imidazolyl propionate are known to alkylate cysteine-46 selectively in liver alcohol dehydrogenase [I -41. X-ray analysis has shown that Cys-46 is one of the three protein ligands to the active-site zinc ,atom [5]. Kinetic investigations have shown that the alkylalion of this residue results from the formation of a reversible complex prior to irreversible alkylation [I]. The reversible complex is formed by carboxylate binding to the general anion-binding site, identified as arginine-47 [5]. This site is part of the coenzyme-binding site, and is where the pyrophosphate group of the coenzyme binds. Several anions thus bind in competition with both coenzyme and a-haloacid, and recently it was shown that even phosphate anions bind to this site [6]. Selective alkylation of Cys-46 with iodoacetate has been taken as a typical example of affinity labelling or active-site-directed 'irreversible inhibition [7].So far, attempts to affinity label liver alcohol dehydrogenase with other haloacids have been unsuccessful, even though slow inactivation has been reported with 2-iodopropionate, 3-bromopropionate, 3-iodopropionate and also with the 4, 5 and 6 carbon bromoacids [8,9]. Interpretation of these inactivation results has suffered from a lack of knowledge of the chemical reactivity of the different haloacids. In a recent study, the alkylating reactivity of a series of alkyl halides was measured by reaction with the free thiolate anion ofcysteine [lo]. The chemical reactivity of the haloacids varied greatly. To understand affinity labelling of liver alcohol clehydrogenase with haloacids, it is therefore of vital importance to compare the enzyme reaction with a model reaction involving the corresponding group free in solution.In this work, iodoacetamide and a series of h...

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“…In three of the compounds the ring is 1 -methylated. Two of the labels have also been available as enantiomers, and the following paper deals with the stereospecific role of configuration [8].…”

Section: Introductionmentioning

confidence: 99%