Steady-state kinetics inhibition studies

Kinetics of O-Methylaiion. The steady state kinetic analysis of these enzymes (41,42) was consistent with a sequential ordered reaction mechanism, in which 5-adenosyl-L-methionine and 5-adenosyl-L-homocysteine were leading reaction partners and included an abortive EQB complex. Furthermore, all the methyltransferases studied exhibited competitive patterns between 5-adenosyl-L-methionine and its product, whereas the other patterns were either noncompetitive or uncompetitive. Whereas the 6-methylating enzyme was severely inhibited by its respective flavonoid substrate at concentrations close to Km, the other enzymes were less affected. The low inhibition constants of 5-adenosyl-L-homocysteine (Table I) suggests that earlier enzymes of the pathway may regulate the rate of synthesis of the final products. 

Pope, A.J. et al. 1998. Characterization of isoleucyl-tRNA synthetase from Staphylococcus aureus II mechanism of inhibition by reaction intermediate and pseudomonic acid analogues studied using transient and steady-state kinetics. J. Biol. Chem. 273, 31691-31701. 

Steady-state kinetic studies showed that the kinetics of the enzyme resemble those of the vanadium bromoperoxidases. The chloroperoxidase exhibits a pH profile similar to vanadium bromoperoxidases although the optimal pH of 4.5-5.0 is at a lower value. At low pH the enzyme is inhibited by chloride in a competitive way whereas at higher pH values the activity displays normal Michaelis-Menten type of behavior (see Michaelis Constant). The log Km for chloride increases linearly with pH whereas that for hydrogen peroxide decreases with pH demonstrating that in the catalytic mechanism protons are involved. These observations have led to a simplified ping-pong type of mechanism for the chloroperoxidase similar to that shown in (Figure 1). 

Steady-state kinetics studies of the inhibition of the enzyme by oxR-mate and oxalate 203) hinted at the existence of enzyme-oxamate complexes. No binding of 0.15 milf oxamate was detected in the ultracentrifuge. However, Siidi 283) reported that oxalate (15 mM) and oxamate (60 mM) protect M4 enzyme from heat denaturation. Oxaloacetate and fructose 1,6-diphosphate are effective at protecting against thermal denaturation at 1 mM concentration 286). It is possible that these anions bind at sites similar to those detected in the dogfish M4 LDH molecule 139). 

Steady-state techniques remain a major mode of enzyme investigation forming the basis of most enzyme assays, inhibition studies, pH profiles, etc. The detailed kinetics of this area are discussed well elsewhere in this volume [1] and our coverage here is selective of the common uses of steady-state kinetics. 

Garcia-AUes, L.F. Zahn, A. Erni, B. Sugar recognition by the glucose and mannose permeases of Escherichia coli. Steady-state kinetics and inhibition studies. Biochemistry, 41, 10077-10086 (2002)... 

Differentiation between reaction mechanisms can be achieved by careful scmtiny of the K versus substrate concentration patterns (Fig. 7.4). The adage that a picture tells a thousand words is quite applicable in this instance. It is difficult to determine the mechanism of an enzyme-catalyzed reaction from steady-state kinetic analysis. The determination of the mechanism of an enzymatic reaction is neither a trivial task nor an easy task. The use of dead-end inhibitors and alternative substrates, study of the patterns of product inhibition, and isotope-exchange experiments... 

Reverse transcriptase (RT) plays a critical role in the early steps of the life of human immunodeficiency virus (HIV) (304), and for over a decade has been one of the major targets of AIDS therapy. Polycitone A (280) was found to be a potent general inhibitor of retroviral reverse transcriptases and cellular DNA polymerases (305). Polycitone A exhibited potent inhibitory capacity of both RNA- and DNA-directed DNA polymerases. It inhibits retroviral reverse transcriptases (RTs) of human immunodeficiency virus type 1 (HIV), murine leukemia virus (MLV) and mouse mammary tumor virus (MMTV)] as efficiently as cellular DNA polymerases of both DNA polymerases a and p and the prokaryotic Klenow fragment of Escherichia coli DNA polymerase I. The mode and mechanism of inhibition of the DNA-polymerase activity associated with HIV-1 RT by polycitone A (280) have been studied. The results suggest that the inhibitory capacity of the DNA polymerase activity is independent of the template-primer used. The RNase H function is hardly affected by this inhibitor. Polycitone A has been shown to interfere with DNA primer extension, as well as with the formation of the RT-DNA complex. Steady-state kinetic studies demonstrate that this inhibitor can be considered as an allosteric inhibitor of HIV-1 RT. The target site on the enzyme may be also spatially related to the... 

The influence of electronegative additives on the CO hydrogenation reaction corresponds mainly to a reduction in the overall catalyst activity.131 This is shown for example in Fig. 2.42 which compares the steady-state methanation activities of Ni, Co, Fe and Ru catalysts relative to their fresh, unpoisoned activities as a function of gas phase H2S concentration. The distribution of the reaction products is also affected, leading to an increase in the relative amount of higher unsaturated hydrocarbons at the expense of methane formation.6 Model kinetic studies of the effect of sulfur on the methanation reaction on Ni(lOO)132,135 and Ru(OOl)133,134 at near atmospheric pressure attribute this behavior to the inhibition effect of sulfur to the dissociative adsorption rate of hydrogen but also to the drastic decrease in the... 

Fromm and Rudolph have discussed the practical limitations on interpreting product inhibition experiments. The table below illustrates the distinctive kinetic patterns observed with bisubstrate enzymes in the absence or presence of abortive complex formation. It should also be noted that the random mechanisms in this table (and in similar tables in other texts) are usually for rapid equilibrium random mechanism schemes. Steady-state random mechanisms will contain squared terms in the product concentrations in the overall rate expression. The presence of these terms would predict nonhnearity in product inhibition studies. This nonlin-earity might not be obvious under standard initial rate protocols, but products that would be competitive in rapid equilibrium systems might appear to be noncompetitive in steady-state random schemes , depending on the relative magnitude of those squared terms. See Abortive Complex... 

Except for very simple systems, initial rate experiments of enzyme-catalyzed reactions are typically run in which the initial velocity is measured at a number of substrate concentrations while keeping all of the other components of the reaction mixture constant. The set of experiments is run again a number of times (typically, at least five) in which the concentration of one of those other components of the reaction mixture has been changed. When the initial rate data is plotted in a linear format (for example, in a double-reciprocal plot, 1/v vx. 1/[S]), a series of lines are obtained, each associated with a different concentration of the other component (for example, another substrate in a multisubstrate reaction, one of the products, an inhibitor or other effector, etc.). The slopes of each of these lines are replotted as a function of the concentration of the other component (e.g., slope vx. [other substrate] in a multisubstrate reaction slope vx. 1/[inhibitor] in an inhibition study etc.). Similar replots may be made with the vertical intercepts of the primary plots. The new slopes, vertical intercepts, and horizontal intercepts of these replots can provide estimates of the kinetic parameters for the system under study. In addition, linearity (or lack of) is a good check on whether the experimental protocols have valid steady-state conditions. Nonlinearity in replot data can often indicate cooperative events, slow binding steps, multiple binding, etc. 

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