Substrate concentration enzymology

Michaelis-Menton equation/kinetics This equation, which is central to enzymology, describes the relationship between the initial rate of reaction (v) and the substrate concentration (Q. It gives the initial rate of reaction as v = V ax C/(K +C) where V ax is the maximum velocity of reaction, C is the concentration of substrate and is the Michaelis-Menton constant. C is equal to the Michaelis-Menton constant when vis 50% of micro- A prefix meaning small. 

As the interrelations between the enzyme activity and a are not linear in all cases, deviations from the activity-pH curves can be expected in the heterogeneous system as compared to homogeneous enzymology. Especially the proportion of sites working at the same external substrate concentration depends on a. Several observations and comments on these problems are cited in references [24, 40, 41, 44, 45, 52] etc. and an illustration is given in Figure 7. 

Selected entries from Methods in Enzymology [vol, page(s)] Theory, 63, 159-162 activation effect, 63, 174, 175 analysis, 63, 140, 159-183 burst, 64, 20, 203, 215 enzyme concentration, 63, 175-177 hysteresis, 64, 197, 200-204 limitations, 63, 181-183 plotting, 63, 177-180 practical methods, 63, 175-177 reversible inhibitor action, 63, 163-175 reversible reaction, 63, 171-175 simulation of, 63, 180 advantages and disadvantages, 249, 61-62 analysis, in kinetic models of inhibition, 249, 168-169 concave-down, 249, 156 concave-up, 249, 156 with enzyme-product complex instability, 249, 88 with enzyme-substrate instabil-... 

Enzymology,29 techniques of isolation, and descriptions of a number of them. Apparently, only three have been considered for preparative chemistry, that is, aldolase, sialyl aldolase, and Kdo synthetase. However, whole cells of some strains of Escherichia coli have been used as sources of fucu-lose 1-phosphate aldolase (E.C. 4.1.2.17) or rhamnulose 1-phosphate aldolase (E.C. 4.1.2.19).30 Extraction, and concentration to a suitable degree of homogeneity, of noncommercially available aldolases are not difficult. The examination of their synthetic possibilities could be very rewarding for we already observe that the wealth of chemicals prepared with the help of aldolase and sialyl aldolase far exceeds what they make in Nature. Still, not any aldehyde, however hydrophilic, is a substrate for aldolases. 

The current method for the hyalurcnidase assay described in the United States Pharmacopeia (USP) [132] is based on the inability of hydrolyzed potassium hyalurooate to form a complex precipitate with proteins from added serum, reflected in a decreased turbidity of the reaction mixture (measured after 30 min). The method is, from the enzymological point of view, not well defined since it does not actually evaluate the kinetics of the hydrolysis of the substrate. An assay with end-point determination is only valid if the reaction rate does not change during this reaction time. We found that only with the two lowest test concentration (0=15 nnH 0=3 PJ) was this condition fulfilled, while with the three higher test concentrations the reaction is not linear. Commercially available hy aluronates can be contaminated with chondroitln sulfates. They are more acidic than... 

In recent years kinetic and mechanistic studies have been done on the copper oxidases. Recent work has concentrated on laccase and tyrosinase, and since these two enzymes are good examples of the complexities involved, the rest of this paper concentrates on the enzymology of these two proteins. They also give rise to interesting comparisons since they have some substrates in common (e.g., catechol) but differ in certain aspects of their physicochemistry and mechanism. 

When one of the substrates is water (i.e., when the process is one of hydrolysis), with the reaction taking place in aqueous solution, only a fraction of the total number of water molecules present participates in the reaction. The small change in the concentration of water has no effect on the rate of reaction and these pseudo-one substrate reactions are described by one-substi ate kinetics. More generally the concentrations of both substrates may be variable, and both may affect the rate of reaction. Among the bisubstrate reactions important in clinical enzymology are the reactions catalyzed by dehydrogenases, in which the second substrate is a specific coenzyme, such as the oxidized or reduced forms of nicotinamide adenine dinucleotide, (NADH), or nicotinamide adenine dinucleotide phosphate, (NADPH), and the amino-group transfers catalyzed by the aminotransferases. 

It is well known that the water content of the reaction medium (i.e., the solvent and solid enzyme-containing phase) has a strong impact on nonaqueous enzymology. Moreover, for a given reaction, enzyme preparation, and medium composition, there is an optimal water content for maximizing the enzyme activity, or the initial rate of reaction. The optimal value is a strong function of the presence and concentration of substrates, and properties of the solid phase. Moreover, the enzyme, immobilization matrix, and continuous phase all compete for adsorption/retention of water molecules. Polar solvents are known to strip away water molecules from solid-phase enzymes. ... 

However, a distinct difference exists in enzymological properties of these two proteases. The protease activity in the juice of A. arguta and its interspecific hybrid fruits is 18-33 times that of Ha)ward, even though actinidin concentration is merely 1.7-3.7 times greater (Nishiyama et al, 2004a Table V). Moreover, a purified protease in Shinzan fruit exhibits decidedly different specificities toward synthetic peptide substrates compared to actinidin purified from Hayward fruit (Nishiyama et al., 2004a Table VI). 

Enzymes are known to be extremely sensitive to their chemical environment and it is for this reason that variations in pH, ionic strength and co-factor or activator concentration are examined during the development of enzyme assays. Since it now appears that many alterations in enzymological activity are brought about with the aid of activity sites on the molecule, other than the sites responsible for substrate binding, an enzyme may be, in fact, a sensor of the intracellular environment capable of translating even small changes in milieu into a variation of its own catalytic activity [19]. 

As indicated above, this kind of experiment is also often used for the determination of the concentration of enzyme active sites by application of equation (S. 1.22). While this has been useful in many cases, it has also led to one major confusion in enzymology in the 1970s. This was the so-called half of sites reactivity hypothesis. It was based on the fact that with some enzymes reactions with certain substrates resulted in burst amplitudes 77 corresponding to approximately half the number of enzyme sites in the solution. It was postulated that in many oligomeric enzymes, in any pair of sites, the states of the two partners alternated, one active and one inactive form. In many cases this has turned out to be due to trivial artefacts, which were summarized by Gutfreund (1975). In some special cases, notably in membrane enzymes, it may be correct. In a group of interesting cases the low burst yield is due to the phenomenon of on enzyme equilibration ... 

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