Alkyl-enzyme

Ring opening of the epoxide and generation of the diol product proceeds by a two-step process. In the first step the epoxide is opened forming an alkylated enzyme, and in the second step the diol product is released and enzyme is regenerated. Mechanistically, the two... 

H. F. Tzeng, L. T. Laughlin, S. Lin, R. N. Armstrong, The Catalytic Mechanism of Microsomal Epoxide Hydrolase Involves Reversible Formation and Rate-Limiting Hydrolysis of the Alkyl-Enzyme Intermediate , J. Am. Chem. Soc. 1996, 118, 9436 -9437. 

ZnSR(TmXyl) (R = alkyl) complexes have been prepared to reproduce the S3Zn-SR coordination environment of thiolate-alkylating enzyme. The most reactive thiolate, ZnSHt(TmXyl)], reacts slowly with trimethyl phosphate yielding methyl-ethylthioether and... 

Figure 40 (continued) The effect of an active-site-directed inhibitor (a) structure of the wild-type enzyme (b) structure of the substrate-bound enzyme and (c) structure of the alkylated enzyme [23]... 

Tzeng H-F, Laughlin LT, Lin S, Armstrong RN. The catalytic mechanism of microsomal epoxide hydrolase involves reversible formation and rate-limiting hydrolysis of the alkyl-enzyme intermediate. J Am Chem Soc 1996 118 9436-9437. 

I Attack of the enolate ion on the iminium cation of W, N -methylene-THF forms a covalent bond between the inhibitor and the coenzyme forming the alkylated enzyme). 

Enzyme samples (150 - 200 mg) were dialyzed extensively after labeling and chromatographed on Whatman CM-52 (2.5 x 25 cm) with 0.1 M phosphate, 0.100 M K, pH 5.7. Without the chromatographic purification step these samples also contained significant amounts of autolyzed forms which, while catalytically inactive, gave distinct spectral peaks in the NMR. Furthermore, the subsequent kinetic experiments required pure Met-192 alkylated enzyme, free of unalkylated native enzyme which was always residual in the alkylation reaction mixture. 

Consequently/ It was not surprising to discover a rate acceleration for the alkylated enzyme derivatives here if the suspected mechanism was similar to that for indole. As controls we examined acetamido (Met-192-CH200NH2)-chymotrypsin, which lacks the aromatic moiety (called acet.-chymotrypsin), acetanilido-chymotrypsin, which lacks the -CF group on the phenyl ring (called H-), and native chymotrypsin. In order to assess the indole (rate accelerating) effects of these derivatives we measured kinetics for all samples in the presence and absence of 10 mM indole. 

The spectrophotometric determination of the reagent equivalents bound to the enzyme is based on the assumption that the spectrum of the alkylated enzyme results from the addition of adenosine deaminase and 9-(p-acetamidobenzyl) adenine spectra. The spectra have two isosbestic points at 244 nm ( = 21.1 X 10 Af- cm" ) and at 265 ( = 21 X 10 cm" ). The adenine derivative exhibits maximum absorbance at... 

The procedure introduced here is based on the assumption that the label released from alkylated enzyme by treatment with a mercaptide ion corresponds numerically to regenerated active sites. Therefore the method allows, in principle, measurement of catalytic activity of a site in a preparation containing inactive protein whether homologous or foreign. The label released can also be used to calculate the equivalent weight of a site when the quantity of the label corresponds to that released on reactivation of enzyme from zero activity to 100% activity. The method is illustrated by description of an actual determination. 

Lohman DC, Edwards DR, Wolfenden R. Catalysis by desolvation the catalytic prowess of SAM-dependent halide-alkylating enzymes. J Hm Chem Soc. 2013 135 14473-14475. 

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