Inhibitors kinetic properties

The basic kinetic properties of this allosteric enzyme are clearly explained by combining Monod s theory and these structural results. The tetrameric enzyme exists in equilibrium between a catalytically active R state and an inactive T state. There is a difference in the tertiary structure of the subunits in these two states, which is closely linked to a difference in the quaternary structure of the molecule. The substrate F6P binds preferentially to the R state, thereby shifting the equilibrium to that state. Since the mechanism is concerted, binding of one F6P to the first subunit provides an additional three subunits in the R state, hence the cooperativity of F6P binding and catalysis. ATP binds to both states, so there is no shift in the equilibrium and hence there is no cooperativity of ATP binding. The inhibitor PEP preferentially binds to the effector binding site of molecules in the T state and as a result the equilibrium is shifted to the inactive state. By contrast the activator ADP preferentially binds to the effector site of molecules in the R state and as a result shifts the equilibrium to the R state with its four available, catalytically competent, active sites per molecule. 

The intrinsic kinetic properties of the victim drug also influence the potential clinical consequences of an interaction. For orally administered medications that undergo significant presystemic extraction, impairment of clearance by a CYP inhibitor may produce increases in bioavailability (reduced presystemic extraction) as a consequence of reduced clearance. The effects may be particularly dramatic for CYP3A substrates (such as triazolam, midazolam, or buspirone) that undergo both hepatic and enteric presystemic extraction. As an example, coadministration of the CYP3A inhibitor ketoconazole with triazolam produced very large increases in area under the plasma concentration-time curve... 

Al. Abrahamson, M., Barrett, A. J., Salvesen, G., and Grubb, A., Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentrations in biological fluids. J. Biol. Chem. 261(24), 11282—11289 (1986). 

Today 11 members of the human PDE superfamily are known, all of which are class I phosphodiesterases and all of which are intracellular or membrane-bound enzymes. Several of the isoenzymes are encoded by more than one gene which, in combination with the presence of different splice variants, brings the number of different PDE proteins to well over 50. The different isoenzymes are characterized according to their substrate specificity, sequence homology, kinetic properties, and sensitivity to certain known PDE inhibitors. Table 9.1 shows these properties together with the predominant tissue expression of the various PDEs. 

Table 2.1. NATURAL INHIBITORS PHYSICAL PROPERTIES AND KINETIC...

Simultaneous to the graph creation, kinetic properties in each vRxn are used to create the appropriate reaction rate equations (ordinary differential equations, ODE). These properties include rate constants (e.g., Michaelis constant, Km, and maximum velocity, Vmax, for enzyme-catalyzed reactions, and k for nonenzymatic reactions), inhibitor constants, A) and modes of inhibition or allosterism. The total set of rate equations and specified initial conditions forms an initial value problem that is solved by a stiff ODE equation solver for the concentrations of all species as a function of time. The constituent transforms for the each virtual enzyme are compiled by carefully culling the literature for data on enzymes known to act on the chemicals and chemical metabolites of interest. 

Dando, C., Schroeder, E. R., Hunsaker, L. A., Deck, L. M., Royer, R. E., Zhou, X., Parmley, S. F., and Vander Jagt, D. L. (2001). The kinetic properties and sensitivities to inhibitors of lactate dehydrogenases (LDH1 and LDH2) from Toxoplasma gondii Comparisons with pLDH from Plasmodium falciparum. Mol. Biochem. Parasitol. 118, 23-32. 

This form may occur in anaerobic tissues, in which lactate is the end product of glycolysis. However, in vitro differences in kinetic properties of the isoenzyme may be inappropriate to explain actual physiological actions for several reasons differences in kinetic properties between LDH-1 and LDH-5 are less marked at 37°C (body temperature) than at 25°C, high intracellular concentrations of the enzyme are present, and differences in the actual ratio of ketopyruvate to enolpyruvate exist (the enol form may be the more potent inhibitor). Furthermore, the occurrence of similar isoenzyme patterns in widely different tissues with divergent metabolic goals (e.g., LDH-5 in liver and musele LDH-1 in heart and erythrocytes) points out our lack of understanding of their preeise role. 

SLPI also contains a methionine residue in its active site (Leu72-Met73), which makes it sensitive to oxidation. Carp and Janoff [105] showed that SLPI could be inactivated by phagocyte-derived oxidants. It was also shown that oxidant-resistant variants (where one to four of the Met residues were replaced by Leu) were better inhibitors of NE when incubated in the presence of the oxidizing agent cis-platinum(II) diamine dichloride [76]. The kinetic properties of SLPI oxidized at the Met residue in position PI demonstrates a 10-fold decrease in the on-rate constant with NE, although this change is less dramatic than in the case of ai-PI [106]. 

Another area of considerable interest is the use of the prodrug concept for better kinetic properties of angiotensin-converting enzyme inhibitors (Sections.2) or fibrinolyticenzymes (70). 

The /3-galactosidase study is an excellent example of the power of site-directed mutagenesis. Huber, Miller, and colleagues prepared and examined five Glu-461-/8-galactosidase substitutions (Asp, Gly, Gin, His, and Lys) (136, 139). All substitutions had /teat values less than 0.3% of the wild-type enzyme except the His-461 mutation, which was approximately 6%. For most of the substitutions it was possible to quantify K, /teat. s. and rates of galactosylation and degalac-tosylation for three substrates, and K values for three inhibitors. Different enzyme kinetic properties resulting from different amino acid substitutions confirm that Glu-461 is directly involved in catalysis and contributes to active site structure stability. Heat inactivation at 55°C occurred more rapidly with each amino acid substitution compared to the wild-type enzyme, except for the structurally conservative Gin substitution, which was only moderately affected. 

Information in support of this hypothesis has been obtained in detailed studies on the inactivation kinetics and mechanism of pig and beef liver MAO-A by A -cyclopropyl-A-arylalkylamines. The structures and some kinetic properties of this second group of MAO inhibitors are summarized in Table III. The inactivation characteristics of the A/-cyclopropylamines are generally similar to those of the propargylamines (1) time-dependent, first-order loss of enzyme activity, saturation kinetics, and protection from inactivation by substrate or product (2) pH-dependent rate of inactivation corresponding to the pH dependence of enzyme activity (3) little activity recovery after exhaustive dialysis (4) partitioning between normal product formation and inactivation and (5) time-dependent conversion of the covalently bound FAD cofactor from the oxidized to a reduced form, which is fairly resistant to reoxidation. An important differ-... 

Zorzano A, Herrera E (1990) Differences in the kinetic properties and sensitivity to inhibitors of human placental, erythrocyte, and major hepatic aldehyde dehydrogenase isoenzymes. Biochem Pharmacol 39 873-878... 

The S. typhimurium ADP-Glc PPase mutants are of some interest because their altered kinetic properties are different than seen for the E. coli allosteric mutants. As seen in Table 3, both activator and inhibitor constants (Ag.s and /0.5) are affected by the mutation in E. coli mutants SG5, 618, and CL1136. In contrast, the... 

Gil, R, M. C. Gonzalvo, A. F. Hernandez, E. Vilanueva, and A. Pla. 1994. Differences in the kinetic properties, effect of calcium and sensitivity in inhibitors of paraoxon hydrolase activity in rat plasma and microsomal fraction of the liver. Biochemical Pharmacology 48 1559-1568. 

H, antagonists are competitive inhibitors with varying pharmacologic and kinetic properties, Ail require hepatic metabolism and cross the placental barrier. 

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