Alternative Substrate Inhibition

The products of enzyme catalysis of a number of the inhibitors were also determined. In some cases, products were determined by analysis of the fluorescence spectmm after exhaustive incubation of enzyme with inhibitor and compared with synthesized standards of possible products. Catalytic products of other benzoxazinones were identified and relative 

Using the enzyme inhibition kinetics and product identification and model studies of alkaline hydrolysis of the compounds, stmcture-activity relationships of the enzyme inhibitor interactions could be understood and predicted. With this knowledge the authors were able to design alternate substrate inhibitors with reasonable chemical stability, inhibition constants in the nanomolar range, and very slow deacylation rates (fcoff), resulting in virtually irreversible inhibition. 

---

MULTISUBSTRATE SYSTEMS. Wong and Hanes were probably among the first to suggest that alternative substrates may be useful in mechanistic studies. Fromm s laboratory was the first to use and extend the theory of alternative substrate inhibition to address specific questions about multisubstrate enzyme kinetic mechanisms. Huang demonstrated the advantages of a constant ratio approach when dealing with alternative substrate kinetics. 

There are three basic methods for carrying out alternative substrate inhibition studies. In the first, the investigator seeks to observe numerical changes in the coefficients of the double-reciprocal form of the enzyme rate expression in the presence and absence of the alternative substrate. For some mechanisms, only certain coefficients will be altered. This method requires extremely accurate estimates of the magnitudes of the coefficients and should always be supplemented with other kinetic probes . 

Whenever using alternative substrate inhibition procedures, the investigator must demonstrate that initial rate conditions remain valid throughout the course of the experiment. This is particularly true of the other sub-strate(s) in multisubstrate enzymes. Because both the substrate under study and its analog are present in the reaction mixtures, the other cosubstrates will be depleted faster. This should always be a consideration in the design of the experiment. 

While requiring the availability of competitive inhibitors for each of the substrates, Fromm s use of competitive inhibitors to distinguish multisubstrate enzyme kinetic pathways represents the most powerful initial rate method. See Alternative Substrate Inhibition... 

De Bruijne, A.W., Vreeburg, H., and Van Steveninck, J. (1985) Alternative-substrate inhibition of L-lactate transport via the monocarboxylate-specific carrier system in human erythrocytes. Biochimica et Biophysica Acta, 812, 841-844. 

The technique of alternate substrate inhibition, which was pioneered by Fromm (38), involves the use of alternate substrates (that produce alternate products) as inhibitors of the formation of the specific product from the variable... 

In this chapter, mechanism-based inhibition is discussed in its broadest sense, where an inhibitor is converted by the enzyme catalytic mechanism to form an enzyme-inhibitor complex. Other terms used in the literature for mechanism-based inhibitors include suicide inhibitors, suicide substrate inhibitors, alternate substrates, substrate inhibitors, and enzyme inactivators, as well as irreversible, catalytic, or cat inhibitors. The terms alternate substrate inhibition and suicide inhibition are used here to describe the two major subclasses of mechanism-based inhibition. 

An alternate substrate inhibitor produces a stable intermediate during the normal course of catalysis, tying up the enzyme in its E-1 form. Although there can be many steps during the process, and more than one product may be formed. Scheme 13.1 shows the essential steps of the mechanism of inhibition. To fully characterize alternate substrate inhibition, the... 

Because continuous assays monitor only free enzyme, they do not distinguish between E-I, E-I, or the E()X complex. Therefore, obs represents the apparent inactivation rate, a combination of inhibition and inactivation. As with alternate substrate inhibition, the second-order apparent inactivation rate can be determined from one of the following equations, depending on whether or not saturation kinetics are observed and the concentration of substrate ... 

---