Enzyme-bound kinetic competence

For nonheme enzymes that fiuther activate dioxygen, it is apparent that the diferrous forms also bind O2 to eventually generate the active species responsible for the oxidative transformations. In the case of MMO, the first intermediate has been labeled compound P (Scheme 2), which subsequently converts to compound Q both are kinetically competent to hydroxylate methane. In the case of RNR, compound X (Scheme 1) is responsible for the one-electron oxidation of a tyrosine residue to generate a tyrosyl radical. Based on chemical considerations and its Mossbauer properties, it has been proposed that compound P is a diferric peroxide species. To date, three model complexes of compound P, with comparable spectroscopic properties, have been structurally characterized (Figure g). In two of these models O2 is bound in a cis... 

Although not a subject of this chapter, Toney and coworkers have quantitated the reaction coordinate of a PLP-dependent L-alanrne racemase [15]. Despite the expectation that the cofactor provides resonance stabilization of the carbanion/enolate anion (quinonoid) intermediate derived by abstraction of the a-proton, the spectroscopic and kinetic analyses for the wild type racemase at steady-state provided no evidence for the intermediate in the reaction catalyzed by the wild type enzyme. Indeed, Toney had previously demonstrated that a kinetically competent quinonoid intermediate accumulates in the impaired R219E mutant [16] Arg 219 is hydrogen-bonded to the pyridine nitrogen of the cofactor. For the wild type racemase, the derived transition state energies for conversion of the bound enantiomers of alanine,... 

A series of rapid chemical quench experiments under single enzyme turnover conditions using radiolabeled S3P or PEP revealed that the tetrahedral ketal phosphate enzyme intermediate was formed as a new peak upon HPLC analysis with detection of the radiolabel. The time course revealed that the formation of the tetrahedral intermediate species paralleled the disappearance of PEP substrate and formation of the EPSP product thus establishing that it was a kinetically competent species. Isolation of the tetrahedral ketal phosphate intermediate using C-2 PEP and S3P as substrates coupled with rapid chemical quench was carried out in conjunction with H-, C-, and P- NMR to provide a definitive structure proof Thus with these studies we have satisfied the criteria for a true reaction intermediate in terms of a chemically plausible mechanism, structure proof, and kinetic competence. Additional studies support the mechanism for EPSP synthase described (Scheme 4, pathway a) including observation of the intermediate bound to the enzyme at internal equilibrium using solution NMR and C-2 PEP as well as using rapid freeze-quench/solid-state NMR studies. ... 

In the assay, the antigen and enzyme-conjugate antigen compete for the antibodies fixed to the tube walls. The free components are removed by washing. Then the enzymatic activity of the bound enzyme is determined. The antigen concentration in the sample Is inversely proportional to the colour intensity. The concentration of the analyte in question Is calculated on a polygonal calibration graph or by calibration with two points. The instrument affords kinetic measurements. 

Inhibitors structurally related to the substrate may be bound to the enzyme active center and compete with the substrate (competitive inhibition). If the inhibitor is not only bound to the enzyme but also to the enzyme-substrate complex, the active center is usually deformed and its function is thus impaired in this case the substrate and the inhibitor do not compete with each other (noncompetitive inhibition). Competitive and noncompetitive inhibition effect the enzyme kinetics differently. A competitive inhibitor does not change but increases. Km (Fig. 25a) in contrast, noncompetitive inhibition results in an unchanged Km and an increased vmax (Fig. 25b). Some enzymes, e.g. invertase, are inhibited by high product concentration (product inhibition). 

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