tailieunhanh - ENZYME KINETICS A MODERN APPROACH – PART 8

Nếu ức chế không phải là cạnh tranh trong tự nhiên, nó không đòi hỏi cơ chế xúc tác và không thể được thay thế chất nền ức chế. Tỷ lệ trên, kon, là tương đương với k1, và tỷ lệ ra, koff, tương đương với tổng của tất cả các con đường của sự cố E-, trong trường hợp này, k-1 + k2. | 160 MECHANISM-BASED INHIBITION T T k1 . k2 E I x x E - I - E X k-1 Scheme various kinetic constants should be determined the reaction products identified and the nature of the inhibition confirmed. If the inhibition is not competitive in nature it does not require the catalytic mechanism and cannot be alternate substrate inhibition. The on rate kon is equivalent to k1 and the off rate koff is equivalent to the sum of all pathways of E-I breakdown in this case k_1 k2. It is possible that multiple products are formed and the rates of formation of these should be included in the koff term. A progress curve or continuous assay is the best way to determine the kon and Ki of an alternate substrate. Addition of an alternate substrate inhibitor to an enzyme assay results in an exponential decrease in rate to some final steadystate turnover of substrate Fig. . In an individual assay both the rate of inhibition kobs and the final steady-state rate C will depend on the concentration of inhibitor. Care must be taken to have a sufficient excess of inhibitor over enzyme concentration present since the inhibitor is consumed during the process. Where possible working at assay conditions well below the Km of the assay substrate simplifies the kinetics as the substrate will not interfere in the inhibition. If the Time Figure . Rate of product formation from an enzymatic reaction with substrate in the presence of an alternate substrate inhibitor showing an exponential decrease in rate to some final steady-state inhibited rate compared to a control rate in the absence of inhibitor. ALTERNATE SUBSTRATE INHIBITION 161 rate of inhibition is too fast to be determined in this fashion saturating or near-saturating concentrations of assay substrate will act as competition for the inhibition reaction and slow the observed rates. The inhibition data are fitted to the following equation for a series of inhibitor concentrations Y Ae-kobst Ct B or Y A 1 - e-kobst Ct B where Y .

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