Alternate substrate inhibition example

These were differently affected by different procedures. For example, when the enzyme was activated at 55°, the increment in ki was slight, but k2 increased 3.5-fold. Similarly, in the presence of EDTA, fc, and k2 values decreased independently, suggesting that the sites for both activities were different. Center and Behai (5) found that with the P. mirabilis enzyme, cyclic 2, 3 -UMP competitively inhibited the hydrolysis of bis(p-nitrophenyl) phosphate. The Ki was 40 pAf very close to the Km for the cyclic nucleotide (Km, 75 yM) which indicated that the two compounds could serve as alternate substrates being hydrolyzed at the same active site. In contrast, 3 -AMP was a mixed inhibitor of cyclic 2, 3 -UMP and bis(p-nitrophenyl) phosphate hydrolysis. Adenosine was a mixed inhibitor of bis(p-nitrophenyl) phosphate hydrolysis but a competitive inhibitor of 3 -AMP hydrolysis. From such kinetic studies Center and Behai (5) suggested that two separate and adjacent sites A and B are involved in the hydrolysis of the diester and phos-phomonoester substrates. Site A serves as a binding site for hydrolysis of ribonucleoside 2, 3 -cyclic phosphates and together with site B catalyzes the hydrolysis of the diester bond. During this reaction 3 -... 

The inhibition of the hexokinase-catalyzed reaction between glucose and ATP by fructose or mannose is an example of competitive inhibition by alternate substrates. Glucose, fructose, and mannose are all substrates of hexokinase and can be converted to product (hexose-6-phosphate). All three hexoses combine with the enzyme at the same active site. Consequently, the utilization of any one of the hexoses is inhibited in the presence of either of the other two. The reaction scheme describing dead-end competitive inhibition is ... 

The previous sections may give the impression that it is an easy matter to establish the mechanism of a multi-substrate enzyme. In fact, more often than not uncertainty and controversy surround such mechanisms for many years despite an abundance of experimental work. We have assumed an ideal situation whereas there are a number of possible obstacles in practice. For example the reaction may be effectively irreversible so that it is only possible to measure the kinetic parameters for one direction of reaction the substrate specificity may be so stringent that it is impossible to apply tests which rely on using a range of alternative substrates the available methods of rate measurement may not be sufficiently sensitive to allow all the kinetic parameters to be determined rehably. The last problem at least is one that allows some hope the kinetic study of NAD-dependent dehydrogenases became much more incisive once fluorescence measurement took over from absorbance measurement as the method of choice [52,57]. Nevertheless there is clearly a need for as many criteria of mechanism as may be mustered and the study of inhibition patterns is a valuable adjunct to the methods already discussed. 

Full kinetic characterization for mechanism-based inhibition can be a challenge. Not only are there multiple rates to determine, but the mechanism of inhibition is often a combination of several different steps. The dividing line between alternate substrate inhibitors and the more eom-plex suicide inhibitors is often blurred, with some alternate substrates being virtually irreversible and some suicide substrates with high partition ratios and a significant alternate substrate element of inhibition. The following examples describe the characterization of an alternate substrate inhibitor and a suicide inhibitor of the serine protease human leukocyte elastase. 

Multiple feedback loops can provide additional fine control. For example, as shown in Figure 9—5, the presence of excess product B decteases the tequitement for substrate 3. Howevet, Sj is also tequited fot synthesis of A, C, and D. Excess B should thetefote also curtail synthesis of all font end products. To circumvent this potential difficulty, each end product typically only partially inhibits catalytic activity. The effect of an excess of two or more end products may be strictly additive or, alternatively, may be greater than their individual effect (cooperative feedback inhibition). 

Many examples of product inhibition are to found. Some dehydrogenases are inhibited by NADH (a co-product of the reaction), e.g. PDH and isocitrate dehydrogenase (ICD), which are involved with the glycolysis and the TCA cycle are two such examples. Hexokinase isoenzymes in muscle (but not liver) and citrate synthase are inhibited by their products, glucose-6-phosphate and citrate respectively offering a very immediate fine tuning of reaction rate to match cellular requirements and possibly allowing their substrates to be used in alternative pathways. 

An experiment with an irreversible inhibitor should carry with it a control experiment involving the addition of a substrate if the location of the reaction with inhibitor is at the active site, then the addition of a substrate will slow down the rate of inhibition. For example, the reactivity of papain (5 pM) with a 1.71 pM solution of 4-toluenesulphonylamidomethyl chloromethyl ketone suffers a drop of 1.68-fold when the substrate (methyl hippurate) is changed from 12.7 to 21.1 mM. The inhibitor which reacts covalently with the enzyme should carry either a radioactive or spectroscopic tag which would enable the location of the altered amino acid to be determined in the sequence, and hence in the three-dimensional X-ray crystallographic map of the enzyme. An alternative approach is to design an inhibitor with groups (analogous to those attached to the substrate) which force it to bind at the active site (Scheme 11.18). 

---