Enzymes total bound

This ratio is of fundamental importance in the relationship between enzyme kinetics and catalysis. In the analysis of the Michaelis-Menten rate law (equation 5.8), the ratio cat/Km is an apparent second-order rate constant and, at low substrate concentrations, only a small fraction of the total enzyme is bound to the substrate and the rate of reaction is proportional to the free enzyme concentration ... 

If we set up the same enzyme assay with a fixed amount of enzyme but vary the substrate concentration we will observe that initial velocity (va) will steadily increase as we increase substrate concentration ([S]) but at very high [S] the va will asymptote towards a maximal value referred to as the Vmax (or maximal velocity). A plot of va versus [S] will yield a hyperbola, that is, v0 will increase until it approaches a maximal value. The initial velocity va is directly proportional to the amount of enzyme—substrate complex (E—S) and accordingly when all the available enzyme (total enzyme or E j) has substrate bound (i.e. E—S = E i -S and the enzyme is completely saturated ) we will observe a maximal initial velocity (Pmax)- The substrate concentration for half-maximal velocity (i.e. the [S] when v0 = Vmax/2) is termed the Km (or the Michaelis—Menten constant). However because va merely asymptotes towards fT ax as we increase [S] it is difficult to accurately determine Vmax or Am by this graphical method. However such accurate determinations can be made based on the Michaelis-Menten equation that describes the relationship between v() and [S],... 

The total bound and unbound enzyme concentration, [E]o, is given by ... 

X 10 moles trypsin per liter fluid volume. To demonstrate the feasibility of using the Ford method to determine the active-site of our immobilized enzyme systems, trypsin CVB-PHEMA-PABS-carbamate was treated in a circulation reactor with NPGB and the titration is Illustrated in Figure 4. The amount of p-nitro-phenol produced by the burst is equal to the amount of the active immobilized trypsin which, for this particular system, turns out to be 31% of the total bound enzyme. Active-site titrations of soluble trypsin were performed according to Chase and Shaw (16), and the active molecules for free trypsin was found to be 70% of the total protein involved. Consequently, the retention of active molecules for the immobilized enzyme was calculated 45%. The specific activity is 17% (Table III) for the same system so the efficiency of the system, based on the actually available active sites, was 38%. Thus, 62% of the initially active trypsin bound has lost its activity upon binding. 

It is obvious that conclusions about the amount of enzyme synthesis could not be based on the amount of cellulolytic activity found in the culture medium since the amount of activity found depended on the time at which measurements were made. On the basis of the hypothesis that the cellulolytic enzymes are bound to the surface of the hyphae and are released to the culture medium only slowly after the growth period, we undertook to investigate the release of the enzymes for, in order to determine if an induction-repression mechanism operates, it is necessary to measure total enzyme activity. 

In the equation above tot is the total concentration of enzyme (total amount, if we are talking about the chemicals dissolved in the same volume of solvent). Like in the case of ligand-receptor, under the chapter on chemical equilibrium, we define it as a sum of free enzyme and enzyme bound in complex [ ]tot = [filfree + [ S]. Of course you want to have as little [ ]free 3S possible. There are also a number of assumptions and simplifications used in developing the relations that help us process the enzyme kinetics data. 

At equilibrium the rates of reaction in the two directions are equal. The maximum rates in each direction, Va and Vb, are obtained when all of the enzyme is bound to one substrate. The rate of reaction in each direction at equilibrium is that fraction of V— given by the actual enzyme-substrate complex divided by total enzyme. 

Soluble proteins make up 25-30% of the total protein in muscle tissue. They consist of ca. 50 components, mostly enzymes and myoglobin (cf. Table 12.5). The high viscosity of the sarcoplasm is derived from a high concentration of solubilized proteins, which can amount to 20-30%. The glycolytic enzymes are bound to the myofibrillar proteins in vivo. 

If one suspects, or better yet knows, that some of the paramagnetic species is not totally bound to the enzyme, then control experiments must be conducted to assess the paramagnetic contribution to l/Tj and l/Tj of nuclei in binary paramagnetic ion-ligand complexes. Without such a correction, overestimates of relaxation rates are obtained that result in underestimates of distances. 

It has often been questioned whether the rates and kinetics of purified enzymes, determined in very dilute solutions with high concentrations of their substrates, but not always of their cofactors, can be extrapolated to the conditions prevailing in the matrix. Much of the mitochondrial water will be bound to protein by hydrogen bonds and electrostatically, but there is also a pool of free water which may only be a fraction of the total water (Gitomer, 1987). The molar concentrations of intermediates of the citrate cycle and of p-oxidation are very low, usually less than those of most enzymes (Srere, 1987 Watmough et al., 1989 Sumegi et al., 1991). The extent to which cofactors and intermediates bind specifically or nonspecifically to enzymes is not known. It is therefore difficult to estimate concentration of these... 

The distribution of PG (PG) as culture medium supernatant, cell-wall associated and cell-bound enzyme was observed in K. marxianus during the time course of growth in 5% glucose medium (Table 2). PG secretion started between 8 and 12 h after inoculation and approximately 90% of total PG was secreted in early stationary phase. PG was not detected intracellularly after 24 h of growth. 

Because mechanism-based inactivation depends on enzyme catalysis, there cannot be more than one molecule of inactivator bound to the enzyme active site. Thus formation of the covalent E-A species cannot result in a stoichiometry of inactivator to enzyme of greater than 1 1. In the case of multimeric enzymes, however, it may not be necessary to covalently modify all of the enzyme active sites within the multi-mer in order to effect total inactivation of the enzyme. In this situation one may observe a stoichiometry of less that 1 1. Under no circumstances, however, can a mechanism-based inactivator display a stoichiometry of greater than 1 1 with the enzyme. 

We start with two mass balance equations that describe the relationships between total, free and bound forms of the enzyme and ligand (inhibitor), respectively ... 

The actual velocity of the reaction depends on how much of the total amount of enzyme is present in the enzyme-substrate (ES) complex. At low substrate concentrations, very little of the enzyme is present as the ES complex—most of it is free enzyme that does not have substrate bound. At very high substrate concentrations, virtually all the enzyme is... 

The total enzyme concentration is the sum of the concentrations of the free and bound forms, E and AE, and the ratio of the latter values depends only on the substrate concentration and the three rate constants in 17.17. From these observations, it follows that for an enzymatically promoted kinetic reaction k,... 

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