Enzyme reaction specificity

Microbial sensors offer a number of assets, namely (a) they are less sensitive to inhibition by solutes and more tolerant to suboptimal pH and temperature values than are enzyme electrodes b) they have longer lifetimes than enzymes and (c) they are less expensive than enzyme electrodes as they require no active enzyme to be isolated. On the other hand, they lag behind enzyme electrodes in a few other respects thus, (a) some have longer response times than their enzyme counterparts b) baseline restoration after measurement typically takes longer and (c) cells contain many enzymes and due care must be exercised to ensure adequate selectivity e.g. by optimizing the storage conditions or using specific enzyme reactions) —some mutant microorganisms lack certain enzymes. 

Their characteristic optical rotatory dispersion or circular-dichroism curves, and their infrared spectra, rich in characteristic frequencies, may be useful. Paper chromatography permits preliminary identification of the glycosyl phosphate or monosaccharide resulting after degradation, and the specific enzymic reactions of these products are widely used to provide additional evidence. 

Intracellular messenger substances can be formed and degraded again in specific enzyme reactions. Via enzymatic pathways, large amoimts of messenger substances can be rapidly created and inactivated again. 

Thus far we have focused on the general principles of catalysis and on introducing some of the kinetic parameters used to describe enzyme action. We now turn to several examples of specific enzyme reaction mechanisms. 

Certain therapeutic effects can be attributed to the inhibition of specific enzymic reactions. The inhibition of cholinesterase (Section 1.06.3), orotidylate pyrophosphorylase (Section 1.06.5) and of dihydrofolate reductase (Section 1.06.6.3) have already been discussed. They illustrate two modes of action, chemical alteration of the enzyme and competition with a substrate for the active site. 

Table 6.1 lists the water-soluble vitamins with their structures and coenzyme forms. Certain portions of the coenzymes are especially important in their biological activities, and they are indicated by arrows. For example, in case of coenzyme A, a thiol ester is formed between its -SH residue and the acyl group being transferred. And in the case of pyridoxal phosphate, its carbonyl residue forms a Schiff base with the amino group of the amino acid that is being decarboxylated. Fat-soluble vitamins (Table 6.2) are also transformed into biologically active substances. However, with the possible exception of vitamin K, these do not operate as prosthetic groups or cosubstrates in specific enzyme reactions. 

Apart from technical considerations, it is important to identify what mechanistic questions can be addressed by the calculations. For example, different possible candidates for an active site base could be compared, or perhaps the stability of various proposed intermediates could be studied. There is a wealth of unanswered questions regarding aspects of specific enzyme reaction mechanisms, and also on the general principles of enzyme catalysis (e.g. what factors or interactions are most important in reducing the activation energy, how the enzyme reaction compares to the equivalent reaction in solution, etc.). Different types of calculation, within the QM/MM framework, may be required to address different types of question, as demonstrated by the variety of applications and approaches described in section 6. Consider what... 

The prediction that enzymes must bind their transition states tightly indicates that compounds, which are structurally similar to the transition state, may also bind tightly. Such transition state analogs have been synthesized for a number of specific enzymic reactions. It is generally found that they do, in fact, bind to their respective target enzymes much more strongly than do the substrates. This... 

The balance of the sulphur cycle requires that reduced sulphur derivatives eventually be reoxidized to sulphate. A number of photosynthetic and chemosynthetic organisms have the ability to utilize reduced sulphur, particularly HgS, as critical electron donors for ATP production, but these pathways are not of enou general importance to consider here. Pathways are known for the production of sulphide from cysteine, and it is also clear that the oxidation of sulphide can occur in animals with production of sulphate and thiosulphate. What is not certain is to just what extent specific enzyme reactions are involved. The nonenzymatic... 

With regard to the action of pressure on specific enzyme reactions in vitro, it would be anticipated from the results with lumine.scence that a dual effect would be encountered, depending upon the temperature of the experiment i.e., the over-all rate should very likely decrease under pressure at temperatures below the optimum, but increase at temperatures above the optimum because of the greater effect at these temperatures on the reversible denaturation of the protein than on the enzyme reaction itself. 

Thermocouples and thermopiles have been used as transduction devices for chemical sensors. These are usually based on a measurement of the heat of reaction and have been coupled to specific enzyme reactions for selectivity. Sensitivity and selectivity of these devices has yet to reach truly satisfactory levels, but there may be specific applications in which they are practical. They do offer the advantage of reduced susceptibility to fouling, compared to electrochemical detection methods. 

---