PH Effects on enzyme catalysis

Michaelis pH function, pH EFFECTS ON ENZYMIC CATALYSIS MICRO-... 

To describe completely the effects of pH changes on enzyme catalysis is an almost impossible task. Many of the amino acid side chains in an enzyme are ionizable, but in environments with polarities different from that of the free solution, their pKa s (Chap. 3) will probably be significantly altered. However, experimentally, it is a simple matter to determine values of steady-state kinetic parameters (Km, Kmax) of an enzyme for various pH conditions. 

Selected entries from Methods in Enzymology [vol, page(s)] Theory, 63, 340-352 measurement, 63, 365 cryosolvent [catalytic effect, 63, 344-346 choice, 63, 341-343 dielectric constant, 63, 354 electrolyte solubility, 63, 355, 356 enzyme stability, 63, 344 pH measurements, 63, 357, 358 preparation, 63, 358-361 viscosity effects, 63, 358] intermediate detection, 63, 349, 350 mixing techniques, 63, 361, 362 rapid reaction techniques, 63, 367-369 temperature control, 63, 363-367 temperature effect on catalysis, 63, 348, 349 temperature effect on enzyme structure, 63, 348. 

In the example given above, the overall effect of pH on enzyme catalysis can be illustrated as follows ... 

Most proteins, including enzymes, are only effective within a narrow pH range, typically from 5 to 9. The pH effect in the rates of enzyme catalysis, illustrated in Figure 14.8, gives rise to a bell-shaped dependence with a maximum close to pH 7.4, the physiological pH. This is due to the presence of ionisable groups in the amino acid chain, namely -NHj+and -CO2H, which must be in appropriate ionisation states in the vicinity of the active site for enzyme catalysis to be observed. Michaelis interpreted the pH effects on the basis of the mechanism... 

The effects of solution pH on enzyme activity can be particularly informative in defining steps in catalysis that are most affected by interactions with inhibitors. Ionization of different groups on the enzyme can be critical in substrate binding (i.e.,... 

Chemical modification of proteins has been extensively studied over the years to identify which amino acids are involved in catalysis. Much less work has been carried out on its influence on enzyme stability. Chemical modification of proteins may yield stabilization, destabilization or no effect at all. Martinek and Berezin (1978) reported the dependence of the thermostability of chymotrypsin on the degree of alkylation of its amino groups up to 30% alkylation the stability rose slightly at 90% substitution stability increased markedly, with a maximum (110-fold) at 95% stability fell to nearly initial values when 100% amino groups were modified. (With these modifications, the optimum pH of the errzyme can change and one must therefore be cautious in comparing two different... 

The ALS isolated as described in Table III displayed typical Michaelis-Menten kinetics with respect to pyruvate with a Km of 2.44 mM. Substrate concentrations as high as 50x Km had no effect on the rate of the reaction. Thiamine pyrophosphate, FAD and Mg(2+) were an absolute requirement for catalysis by the purified enzyme. These properties are consistent with observations made by others (30). Optimum activity was obtained at pH 7.1 and 37C, which were also the best conditions for inhibition by TP. There was no significant difference in the 1(50) value of TP whether ALS was taken after step 2 or 5, indicating low potential for non-specific binding of the herbicide to other proteins. 

Effects of Temperature and pH on Enzyme-Catalyzed Reactions Detailed Mechanisms of Enzyme Catalysis... 

The mysterious behaviour of bio-macromolecules is one of the outstanding problems of molecular biology. The folding of proteins and the replication of DNA transcend all classical mechanisms. At this stage, non-local interaction within such holistic molecules appears as the only reasonable explanation of these phenomena. It is important to note that, whereas proteins are made up of many partially holistic amino-acid units, DNA consists of essentially two complementary strands. Nonlocal interaction in DNA is therefore seen as more prominent, than for proteins. Non-local effects in proteins are sufficient to ensure concerted response to the polarity and pH of suspension media, and hence to direct tertiary folding. The induced fit of substrates to catalytic enzymes could be promoted in the same way. Future analysis of enzyme catalysis, allosteric effects and protein folding should therefore be, more ambitiously, based on an understanding of molecular shape as a quantum potential response. The function of DNA depends even more critically on non-local effects. 

The second reason pAf values are perturbed in log( V) profiles is that there has been a shift in rate-limiting steps. For example, with malic enzyme the pAT values of 6 and 8 seen in the VIK profile for malate are shifted outward in the log(F) profile by approximately 1.3 pH units because at neutral pH NADPH release from E-NADPH, rather than catalysis, is the rate-limiting step for V. Only when incorrect protonation has slowed catalysis by a factor of about 20 does it become equal to the rate of NADPH release, and thus the pAf values are perturbed outward by log 20, or 1.3, pH units (81). The deuterium isotope effects on VIK of 1.5 and of 1.0 on V at neutral pH support this interpretation [the isotope effect... 

Breaker and Joyce [16] accomplished the selection of a DNA enzyme that could specifically cleave the phosphodiester bond of a ribonucleotide. The DNA pool synthesized for this purpose contained a single ribonucleotide at a specific position within a primer binding site, in order to avoid the possible effect of RNA on the catalysis. In addition, they assumed that a DNA-dependent cleavage at pH 7 would require a cofactor, possibly a... 

After optimizing the assay conditions, including ionic strength, pH, temperature, activator (Ca ) concentration, and polymer concentration, a calibration curve was developed, which allows the lipid substrate concentration to be determined from the fluorescence intensity. The calibration curve allows the enzyme catalysis kinetics parameters (e.g.. Km and Vmax) to be measured. This PLC turn-off assay is effectively inhibited by known inhibitors (F and EDTA), which demonstrates that the sensor relies on the specific catalysis reaction by PLC. It has been demonstrated to be a sensitive (detection limit 0.5nM enzyme concentration), fast (<5 min), and selective (good specificity over phospholipase A and D, and other nonspecific proteins) PLC assay, which can be carried out at very low initial substrate concentration (in the range of micromolar to nanomolar). 

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