Enzyme catalytic constant

Catalytic Constants and Activation Parameters for the Enzymic Hydrolysis of /i-D-Glucopyranosides ... 

KDHRF A homologous restriction factor binds to C8 65KDHRF A homologous restriction factor, also known as C8 binding protein interferes with cell membrane pore-formation by C5b-C8 complex Kcat Catalytic constant a measure of the catalytic potential of an enzyme Ka Equilibrium dissociation constant kD Kilodalton Kd Dissociation constant KD Kallidin... 

Different from conventional chemical kinetics, the rates in biochemical reactions networks are usually saturable hyperbolic functions. For an increasing substrate concentration, the rate increases only up to a maximal rate Vm, determined by the turnover number fccat = k2 and the total amount of enzyme Ej. The turnover number ca( measures the number of catalytic events per seconds per enzyme, which can be more than 1000 substrate molecules per second for a large number of enzymes. The constant Km is a measure of the affinity of the enzyme for the substrate, and corresponds to the concentration of S at which the reaction rate equals half the maximal rate. For S most active sites are not occupied. For S >> Km, there is an excess of substrate, that is, the active sites of the enzymes are saturated with substrate. The ratio kc.AJ Km is a measure for the efficiency of an enzyme. In the extreme case, almost every collision between substrate and enzyme leads to product formation (low Km, high fccat). In this case the enzyme is limited by diffusion only, with an upper limit of cat /Km 108 — 109M. v 1. The ratio kc.MJKm can be used to test the rapid... 

The following diagram illustrates the reaction of acetylcholinesterase (E) with acetylcholine (S) to produce an acetylenzyme Intermediate (E ) with later hydrolysis of the Intermediate and regeneration of the enzyme.% Is calculated as shown in the diagram. The catalytic constant, kcat, refers to the overall decomposition of the enzyme Intermediate (E.S, E ). 

Values of a catalytic constant, 27 k computed from (6) are listed in Table V. It is obvious that the imidazole-substituted dodecylpoly-enimines are more than 100 times as effective as the simple imidazole28,29 molecule itself. The catalytic constant for the imidazole-dodecyl polymer in fact approaches that of the enzyme chymotrypsin. 

What is the exact influence of water and organic molecules on the enzyme structure Could its effects on properties such as selectivity, affinity, binding constants, and catalytic constants be predictable by controlling the hydration/solvation state ... 

It is also feasible that, following changes in the value of Kmax under different reaction conditions, it might be possible to obtain information concerning the kinetics of the rate-limiting step in the decomposition of ES. The catalytic constant or turnover number ( <, ) is a first-order rate constant that refers to the properties and reactions of the enzyme-substrate, enzyme-intermediate, and enzyme-product complexes. The units of kca, are time , and l/k t is the time required to turn over a molecule of substrate on an active site. 

For a-chymotrypsin, the procedure of active-site titration for the calculation of active enzyme concentration and thus of the catalytic constant kcat is long established. The original active-site titration experiment on a-CT by Hartley and Kilby (Hartley, 1954) was performed with ethyl p-nitrobenzoate (Figure 9.2). 

Another way of evaluating enzymatic activity is by comparing k2 values. This first-order rate constant reflects the capacity of the enzyme-substrate complex ES to form the product P. Confusingly, k2 is also known as the catalytic constant and is sometimes written as kcal. It is in fact the equivalent of the enzyme s TOF, since it defines the number of catalytic cycles the enzyme can undergo in one time unit. The k2 (or kcat) value is obtained from the initial reaction rate, and thus pertains to the rate at high substrate concentrations. Some enzymes are so fast and so selective that their k2/Km ratio approaches molecular diffusion rates (108—109 m s-1). This means that every substrate/enzyme collision is fruitful, and the reaction rate is limited only by how fast the substrate molecules diffuse to the enzyme. Such enzymes are called kinetically perfect enzymes [26],... 

Determine the Michaelis constant Km and the catalytic constant k3 for the enzyme under these conditions. 

Synthetic substrates allow rapid determination of the catalytic constants of an enzyme. Nevertheless, it is known that the environment of the peptide bond depends largely on physico-chemical conditions of the applied media, and imposed steric hindrance. Since these parameters are important, the hydrolysis of purified (3-casein was studied at different pHs. The kinetic analysis revealed that the mutant conserved the native trypsin capacity to hydrolyze peptide bonds containing arginyl and lysyl residues. The optimal pH of activity changed considerably according to the mutation. 

The most commonly used unit of enzyme activity has been defined as the amount of activity that catalyzes the transformation of 1 (xmol of substrate per minute under specified assay conditions. Specific activity is the number of enzyme units per milligram of protein ((imol/min per milligram of protein). The turnover number or catalytic constant is equal to the units of enzyme activity per nmole of enzyme ((xmol/min per nmol of enzyme). 

Though the values of the Michaels complex (Km 10 4 - 10 5 M) for reactions catalyzed by natural enzymes and catalytic antibodies were found to be of the same order of magnitude, the catalytic constants (kc,) for CAs commonly 104 fold lower than that for correspondent enzymes. Experimental ratio values (kcat/lWat) for CAs ranges within 102-106, while these values for similar enzymatic reactions can reach 1017... 

In the simple Michaelis-Menten mechanism described by Equation 17.9, there is only one E. S complex and all binding steps are rapid. In this instance, is the product of the enzyme concentration [E] and k, (also known as Aicat) which is the first-order rate constant for the chemical conversion of the E. S complex to free enzyme and product. The catalytic constant is often referred to as the turnover number because it represents the maximum number of substrate molecules converted to products per active site per unit time. In a more complicated reaction, k at is a function of all the first-order rate constants and, effectively, sets a lower limit on all the chemical rate constants. 

Hwang et al.131 were the first to calculate the contribution of tunneling and other nuclear quantum effects to enzyme catalysis. Since then, and in particular in the past few years, there has been a significant increase in simulations of QM-nuclear effects in enzyme reactions. The approaches used range from the quantized classical path (QCP) (e.g., Refs. 4,57,136), the centroid path integral approach,137,138 and vibrational TS theory,139 to the molecular dynamics with quantum transition (MDQT) surface hopping method.140 Most studies did not yet examine the reference water reaction, and thus could only evaluate the QM contribution to the enzyme rate constant, rather than the corresponding catalytic effect. However, studies that explored the actual catalytic contributions (e.g., Refs. 4,57,136) concluded that the QM contributions are similar for the reaction in the enzyme and in solution, and thus, do not contribute to catalysis. 

The kinetic investigation of enzymic reactions and suitable non-enzymic standard reactions permits, as just described, the numerical calculation of measures of enzyme catalytic power. These measures are ratios of rate constants and they correspond to equilibrium constants for reactions of free enzyme or enzyme complexes with the transition state for the standard reaction to generate complexes of the enzyme with various transition states along the enzymic reaction pathway, sometimes with liberation of other ligands (see the examples above). 

Novel kinetic formulations. The description given above of the calculation of measures of enzyme catalytic power relies initially on empirically determined rate constants for enzymic and non-enzymic reactions. The numerical results at the initial stage are therefore theory-free and may be used for many purposes with perfect confidence. 

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