Các giá trị của độ dốc và đánh chặn có thể dễ dàng thu được bằng cách sử dụng hồi quy tuyến tính. Như vậy, từ một đường cong tiến bộ duy nhất (tức là, một St-t tập dữ liệu), nó có thể để có được ước tính của Km và kcat. Nếu thủ tục này nghe có vẻ quá tốt là đúng, nó có thể được. Vấn đề chính với thủ tục này là các điều kiện sau đây phải được đáp ứng: | 60 CHARACTERIZATION OF ENZYME ACTIVITY A plot of t 1 ln S0 St versus So St t yields a straight line with slope 1 Km x-intercept V max and y-intercept Vmax Km Fig. . The values of the slope and intercept can readily be obtained using linear regression. Thus from a single progress curve . a single St -1 data set it is possible to obtain estimates of Km and kcat. If this procedure sounds too good to be true it probably is. The major problem with this procedure is that the following conditions must be met 1. The enzyme must be stable during the time course of the measurements used in the determination of reaction velocity. 2. The reverse reaction product to substrate must be negligible. 3. The product must not be inhibitory to enzyme activity. If these conditions are not met particularly the first one this procedure is not valid. Enzyme destabilization reaction reversibility and product inhibition considerations can be incorporated into the kinetic model however this procedure is complex and the validity of the results obtained can be questionable. CHAPTER 4 REVERSIBLE ENZYME INHIBITION An inhibitor is a compound that decreases the rate of an enzyme-catalyzed reaction. Moreover this inhibition can be reversible or irreversible. Reversible enzyme inhibition can be competitive uncompetitive or linear mixed type each affecting Ks and vmax in a specific fashion. In this chapter each type of reversible inhibition is discussed in turn. This is followed by two examples of strategies used to determine the nature of the inhibition as well as to obtain estimates of the enzyme-inhibitor dissociation constant Kj . COMPETITIVE INHIBITION In this type of reversible inhibition a compound competes with an enzyme s substrate for binding to the active site E S - - ES E P 1 IIK EI This results in an apparent increase in the enzyme-substrate dissociation constant Ks . an apparent decrease in the affinity of enzyme for 61 62 REVERSIBLE ENZYME INHIBITION substrate without
