Enzymes, inhibition, substrate turnover number

Acetylcholinesterase is subject to substrate Inhibition at high concentrations, but Mlchaells kinetics are observed at lower concentrations, because the substrate constant and the Mlchaells constant differ by a factor of 100. Turnover numbers run about 2-9 x 10 min l, and (Mlchaells constant) values are about 0.2 mM.76,116 Whatever the source, the enzyme is subject to inhibition by the same reversible and irreversible inhibitors. Most of the kinetic work has been done with the saline-extracted 11S enzyme from electric eel and the detergent-extracted 6S enzyme from erythrocytes. The former Is a tetramer derived from the native enzyme by the action of proteases the latter is a dimer. 

In contrast to the AChR, AChE does not bind bungarotoxin or sulfhydryl reagents. It is inhibited by excess substrate (3 x I0 M), and the of electric eel AChE is about 10 M. The specific activity of the enzyme is one of the highest known 750 nmol/mg-hr, with a turnover time of 30-60 msec and a turnover number of 2-3 x 106. It is therefore one of the most efficient and fastest enzymes known. 

The total activity of a particular enzyme in vivo is determined primarily by three factors (1) the inherent catalytic competence of the enzyme (turnover number moles of substrate transformed per mole of enzyme per unit time) (2) the level of that enzyme which is expressed in relevant tissues (3) the possible presence of agents that inhibit enzyme activity by competitive or non-competitive actions on the enzyme protein. 

The model was then extended to two different dextransucrases from L. mesen-teroides 1299, a soluble and an immobilized form. The parameter sets for the soluble form fitted well with those obtained from the B-512F enzyme. The immobilized form showed a good agreement only for parameters linked to the sucrose concentration (turnover number, A"M. and substrate inhibition constant), but significant differences occurred with other parameters [26]. 

In noncompetitive inhibition, which also is reversible, the inhibitor and substrate can bind simultaneously to an enzyme molecule at different binding sites (see Figure 8.16). A noncompetitive inhibitor acts by decreasing the turnover number rather than by diminishing the proportion of enzyme molecules that are bound to substrate. Noncompetitive inhibition, in contrast with competitive inhibition, cannot be overcome by increasing the substrate concentration. A more complex pattern, called mixed inhibition, is produced when a single inhibitor both hinders the binding of substrate and decreases the turnover number of the enzyme. 

Specific small molecules or ions can inhibit even nonallosteric enzymes. In irreversible inhibition, the inhibitor is covalently linked to the enzyme or bound so tightly that its dissociation from the enzyme is very slow. Covalent inhibitors provide a means of mapping the enzyme s active site. In contrast, reversible inhibition is characterized by a rapid equilibrium between enzyme and inhibitor. A competitive inhibitor prevents the substrate from binding to the active site. It reduces the reaction velocity by diminishing the proportion of enzyme molecules that are bound to substrate. In noncompetitive inhibition, the inhibitor decreases the turnover number. Competitive inhibition can be distinguished from noncompetitive inhibition by determining whether the inhibition can be overcome by raising the substrate concentration. 

Taken together, these experiments indicate that human recombinant cytochromes expressed in E. coli are characterized by substrate specificities, reaction rates, turnover numbers and specific inhibition profiles in keeping with the counterpart enzymes from human liver microsomes. 

Substrate specificity of the purified indoleamine 2,3-dioxygenase from rabbit intestine was examined spectrophotometrically at 24°C. The spectra of the reaction products in either the absence or presence of formamidase were compared with those of authentic compounds. A single enzyme protein catalyzed the oxygenative ring cleavage of d- and L-tryptophan, 5-hydroxy-D- and -L-tryptophan, tryptamine, and serotonin (10). The maximal turnover number was obtained with L-tryptophan (99 mol min -mor of enzyme at 24°C), and the lowest value was with 5-hydroxy-L-tryp-tophan (20 p.Af). A marked substrate inhibition is observed by the L isomers of tryptophan and 5-hydroxytryptophan above 0.2 and 0.06 mM, at pH 6.6, respectively. The compounds including skatole, indole, in-doleacetic acid, 5-hydroxyindoleacetic acid, N-acetyltryptophan, melatonin, and a-methyl-DL-tryptophan, are all inert as substrate. 

The /f -values of enzymes for their substrate are between 10 to 10 M, and their turnover number (number of substrate molecules converted per active site) is of the order of a few thousand per second, but can reach 600000 s (carboanhydrase). Enzymes not only recognize and convert their substrates, but they can also be reversibly inhibited by substrate-simulating molecules and irreversibly by substances that modify functional groups. They can also be affected in their activity by substances that influence their conformation (special effectors, cations, or protons). 

H139L <4> (<4>, mutant enzyme with diminished kinase activity and ATPase activity 150fold greater than that of the wild-type enzyme [12]) [12] H202L <4> (<4>, Km-value for L-homoserine and ATP remain unchanged, the K -value for substrate inhibition by L-homoserine increases about 8fold, the turnover-number decreases by 50%, unlike the wild-type enzyme the L-homoserine ethyl, isopropyl, and n-propyl esters show substrate inhibition [12]) [12]... 

Bull seminal-plasm a hyaluronidase has been purified 180-fold by affinity chromatography on concanavalin A-Sepharose , heparin-Sepharose , and chromatography on Sephadex G-200 and Sephacryl S-200. With hyaluronic acid as the substrate, the specific activity and turnover number of purified hyaluronidase were 3.63 /Umol min per mg (10400 National Formulary units mg of protein) and 214min (mol of product formed per mol of enzyme per min), respectively. Polyacrylamide gel electrophoresis indicated that the purified enzyme migrated as a single band at pH 4.3 and 5.3. Bull seminal-plasma hyaluronidase was markedly inhibited by hydroxylamine, phenyl-hydrazine, and semicarbazide. 

The Michaelis-Menten mechanism of enzyme activity models the enzyme with one active site that, weakly and reversibly, binds a substrate in homogeneous solution. It is a three-step mechanism. The first and second steps are the reversible formation of the enzyme-substrate complex (ES). The third step is the decay of the complex into the product. The steady-state approximation is applied to the concentration of the intermediate (ES) and its use simplifies the derivation of the final rate expression. However, the justification for the use of the approximation with this mechanism is suspect, in that both rate constants for the reversible steps may not be as large, in comparison to the rate constant for the decay to products, as they need to be for the approximation to be valid. The simplest form of the mechanism applies only when A h 2> k. Neverthele.ss, the form of the rate equation obtained does seem to match the principal experimental features of enzyme-catalyzed reactions it explains why there is a maximum in the reaction rate and provides a mechanistic understanding of the turnover number. The model may be expanded to include multisubstrate reaction rate and provides a mechanistic understanding of the turnover number. The model may be expanded to include multisubstrate reactions and inhibition. 

It is a monomeric protein of M.W. about 70,000, shows Kj, values for L-tryptophan and dimethylallyl pyrophosphate of 0.067 and 0.2 mM, respectively, and seems to have a relatively low turnover number, about 7 sec . During studies on this enzyme it was observed (13) that agroclavlne and elymoclavine, the terminal alkaloids in the strain used for the isolation of the enzyme, inhibited purified DMAT synthetase. At concentrations of 3 mM ( v<750 mg/1) agroclavlne and elymoclavine inhibited the enzyme 90% and 70%, respectively. The inhibition is of a mixed or uncompetitive type as shown by kinetic analysis with either tryptophan or dimethylallyl pyrophosphate as the variable substrate (Fig. 6). Subsequently, feedback Inhibition by elymoclavine was also demonstrated by GrSger s group (3) for chanoclavlne cyclase and by us for anthranllate synthetase from... 

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