Physical and enzymatic properties of a class III isozyme of human liver alcohol dehydrogenase: .chi.-ADH

Wagner, Fred; Parés, Xavier; Holmquist, Barton; Vallée, Bert L.

doi:10.1021/bi00305a014

Cited by 117 publications

(109 citation statements)

References 26 publications

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“…Inductively coupled plasma emission spectroscopy analyses revealed that AtzB retained 40 to 70% of its metal content after chelator treatment, suggesting that the 1,10-phenanthroline inactivation was not due to metal removal. Wagner, et al observed similar results with human liver alcohol dehydrogenase, concluding that an enzyme-metal-chelator complex was formed which inhibited activity by blocking substrate access to the active site (39).…”

Section: Resultsmentioning

confidence: 70%

Hydroxyatrazine N -Ethylaminohydrolase (AtzB): an Amidohydrolase Superfamily Enzyme Catalyzing Deamination and Dechlorination

et al. 2007

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Hydroxyatrazine [2-(N-ethylamino)-4-hydroxy-6-(N-isopropylamino)-1,3,5-triazine] N-ethylaminohydrolase(AtzB) is the sole enzyme known to catalyze the hydrolytic conversion of hydroxyatrazine to N-isopropylammelide. AtzB, therefore, serves as the point of intersection of multiple s-triazine biodegradative pathways and is completely essential for microbial growth on s-triazine herbicides. Here, atzB was cloned from Pseudomonas sp. strain ADP and its product was purified to homogeneity and characterized. AtzB was found to be dimeric, with subunit and holoenzyme molecular masses of 52 kDa and 105 kDa, respectively. The k cat and K m of AtzB with hydroxyatrazine as a substrate were 3 s ؊1 and 20 M, respectively. Purified AtzB had a 1:1 zinc-to-subunit stoichiometry. Sequence analysis revealed that AtzB contained the conserved mononuclear amidohydrolase superfamily active-site residues His74, His76, His245, Glu248, His280, and Asp331. An intensive in vitro investigation into the substrate specificity of AtzB revealed that 20 of the 51 compounds tested were substrates for AtzB; this allowed for the identification of specific substrate structural features required for catalysis. Substrates required a monohydroxylated s-triazine ring with a minimum of one primary or secondary amine substituent and either a chloride or amine leaving group. AtzB catalyzed both deamination and dechlorination reactions with rates within a range of one order of magnitude. This differs from AtzA and TrzN, which do not catalyze deamination reactions, and AtzC, which is not known to catalyze dechlorination reactions.

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Section: Resultsmentioning

confidence: 70%

Hydroxyatrazine N -Ethylaminohydrolase (AtzB): an Amidohydrolase Superfamily Enzyme Catalyzing Deamination and Dechlorination

et al. 2007

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“…The moderate inhibition of Mpr activity by EDTA alone and the marginal inhibition by 1,10-phenanthroline suggest that the metal-binding site on the enzyme has a sufficiently high stability constant to render the metal inaccessible to chelating agents. It is noteworthy that others have reported examples of chelating agents being unable to remove the metal ion from some metalloenzymes (4,27).…”

Section: Discussionmentioning

confidence: 99%

Isolation and characterization of a novel extracellular metalloprotease from Bacillus subtilis

et al. 1990

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We have isolated and characterized two minor extracellular proteases from culture supernatants of a strain of Bacillus subtilis containing deletion mutations of the genes for the extracellular proteases subtilisin (apr) and neutral protease (npr) and a minor extracellular protease (epr) as well as intracellular serine protease-I (isp-1). Characterization studies have revealed that one of these enzymes is the previously described protease bacillopeptidase F. The second enzyme, the subject of this report, is a novel metalloprotease, which we designate Mpr. Mpr is a unique metalloprotease that has been purified to apparent homogeneity by using both conventional and high-performance liquid chromatography procedures. Mpr has a molecular mass of -28 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a basic isoelectric point of 8.7. The enzyme showed maximal activity against azocoll at pH 7.5 and 50°C. Mpr was inhibited by dithiothreitol and a combination of j8-mercaptoethanol and EDTA. Activity was moderately inhibited by I-mercaptoethanol and EDTA alone as well as by cysteine and citrate and only marginally by phosphoramidon 1,10-phenanthroline and N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine. Mpr was not inhibited by phenylmethylsulfonyl fluoride. In addition, Mpr showed esterolytic but not collagenolytic activities. Our studies suggest that Mpr is a secreted metalloprotease containing cysteine residues that are required for maximal activity.Sporulation in Bacillus subtilis is a tightly regulated developmental process involving alterations in protein synthesis and intermediary metabolism (5,6,20). At the end of exponential growth and paralleling the onset of sporulation, there is increased production and secretion of extracellular proteases.Two prominent proteases produced during the onset of sporulation are the alkaline serine protease or subtilisin (12, 22) and neutral (metallo-) protease (23)(24)(25). The combined activities of these two enzymes account for 96 to 98% of the total protease activity present in culture supernatants of wild-type sporulating cells (8). Efforts to clone and delete the genes coding for subtilisin (8, 21) and neutral protease (28) have made possible the study of minor proteases (1,13,14) that contribute to the residual activity present in double mutants of B. subtilis incapable of producing either protease. For example, work in our laboratory has identified and resulted in the cloning of a minor extracellular protease gene (epr; 17) from a strain of B. subtilis deleted for the subtilisin (apr) and neutral protease (npr) genes.

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“…The horse E subunit is also included since it is the model structure. Both rat and human class I11 enzymes have a greater molar optical absorbance at 280 nm than the class I enzymes, 77.6 x lo3 M -cm-', versus 48.4 x lo3 M-' cm-for the human forms [27].…”

Section: Correlation Of Substitutions With Changes In Physicochemicalmentioning

confidence: 90%

“…2) and Ser in class I1 appears to be of special interest in this respect. Thus, class I enzymes from both rat and human liver [31, 321 have a significant activity at pH 7.5 as compared to pH 10.0, whereas the class I1 [15,33] and class I11 [17,27] enzymes have considerably less activity. Of the enzymes characterized, all with high activity at pH 7.5 have His-51 (liganding to the coenzyme [5,34]), whereas those with low activity do not have His at position 51.…”

Section: Coenzyme-binding Region and The Segment Involved In Subunitmentioning

confidence: 99%

Rat liver alcohol dehydrogenase of class III

Julià

Parés

Jörnvall

1988

European Journal of Biochemistry

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The amino acid sequence of alcohol dehydrogenase of class I11 from rat liver (the enzyme ADH-2) has been determined. This type of structure is quite different from those of both the class I and the class I1 alcohol dehydrogenases. The rat class I11 structure differs from the rat and human class I structures by 133 -138 residues (exact value depending on species and isozyme type); and from that of human class I1 by 132 residues. In contrast, the rat/human species difference within the class I11 enzymes is only 21 residues. The protein was carboxymethylated with iod0[2~~C]acetate, and cleaved with CNBr and proteolytic enzymes. Peptides purified by exclusion chromatography and reverse-phase high-performance liquid chromatography were analyzed by degradation with a gas-phase sequencer and with the manual 4-N,N-dimethylaminoazobenzene-4'-isothiocyanate double-coupling method.The protein chain has 373 residues with a blocked N terminus. No evidence was obtained for heterogeneity. The rat ADH-2 enzyme of class I11 contains an insertion of Cys at position 60 in relation to the class I enzymes, while the latter alcohol dehydrogenase in rat (ADH-3) has another Cys insertion (at position 111) relative to ADH-2. The structure deduced explains the characteristic differences of the class I11 alcohol dehydrogenase in relation to the other classes of alcohol dehydrogenase, including a high absorbance, an anodic electrophoretic mobility and special kinetic properties. The main amino acid substitutions are found in the catalytic domain and in the subunit interacting segments of the coenzyme-binding domain, the latter explaining the lack of hybrid dimers between subunits of different classes. Several substitutions provide an enlarged and more hydrophilic substrate-binding pocket, which appears compatible with a higher water content in the pocket and hence could possibly explain the higher K , for all substrates as compared with the corresponding values for the class I enzymes. Finally the class I11 structure supports evolutionary relationships suggesting that the three classes constitute clearly separate enzymes within the group of mammalian zinc-containing alcohol dehydrogenases.Mammalian alcohol dehydrogenases (ADH) are zinc-containing enzymes with known relationships to similar enzymes in yeast, plants and other organisms [l -31. The horse enzyme constitutes the crystallographically investigated model enzyme [4] (recent review in [5]). The human enzymes form a complex system of three classes [6], which have all recently been characterized (summaries in [7 -91).

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Physical and enzymatic properties of a class III isozyme of human liver alcohol dehydrogenase: .chi.-ADH

Cited by 117 publications

References 26 publications

Hydroxyatrazine N -Ethylaminohydrolase (AtzB): an Amidohydrolase Superfamily Enzyme Catalyzing Deamination and Dechlorination

Hydroxyatrazine N -Ethylaminohydrolase (AtzB): an Amidohydrolase Superfamily Enzyme Catalyzing Deamination and Dechlorination

Isolation and characterization of a novel extracellular metalloprotease from Bacillus subtilis

Rat liver alcohol dehydrogenase of class III

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