Alternative Substrates

Any compounds (other than the physiologically relevant substrate) that can serve as substrates for a particular enzyme. Alternative substrates compete with the natural substrate and with each other for access to the enzyme's active site. Thus, if one is utilizing an assay that measures the production of the true substrate, then the presence of the alternative substrate will result in competitive inhibition relative to the true substrate.

GENERAL PROPERTIES. Because enzymes rarely exhibit absolute substrate specificity, their use of alternative substrates affords the opportunity to investigate the nature of enzymic catalysis under conditions where the reaction's kinetic and thermodynamic parameter may reveal more about rate-limiting steps or other mechanistic aspects. An excellent example of an enzyme with many alternative substrates is bovine brain hexokinase (Table I).

Table I

Substrate Specificity of Bovine Brain Hexokinase

Table I

Substrate Specificity of Bovine Brain Hexokinase

Michaelis

Relative

Substrate

Constant

Rate

Glucose

0.05 mM

1.0

Fructose

1.6 mM

1.5

Mannose

0.07 mM

0.4

2-Deoxyglucose

0.27 mM

1.0

1,5-Anhydro-D-glucitola

10 mM

1.0

Glucosamine

0.8 mM

0.6

Galactose

100 mM

0.02

aNote: This cyclic ether analogue of glucose is conformationally analogous to the/3-D-glucopyranose, except it lacks the C-1 hydroxyl group.

aNote: This cyclic ether analogue of glucose is conformationally analogous to the/3-D-glucopyranose, except it lacks the C-1 hydroxyl group.

MULTISUBSTRATE SYSTEMS. Wong and Hanes12 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 mechanisms3-5. Huang demonstrated the advantages of a constant ratio approach6-8 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 altered2. This method requires extremely accurate estimates of the magnitudes of the coefficients and should always be supplemented with other kinetic probes6.

In the second and most commonly used method, the investigator studies the alterations in the patterns of the initial rate data (usually graphically presented as double-reciprocal plots). In these studies of multisubstrate and multiproduct enzyme-catalyzed reactions, an investigator can measure the rate of the reaction by either by observing any increase in the concentration of a common

Amadori Rearrangement product (i.e., a product formed by both the normal substrate and the alternative substrate) or by detecting an increase in the concentration of the product not formed from the alternative substrate. Each procedure results in its own characteristic set of rate expressions and will produce different patterns in the double-reciprocal plots (1/v vs. 1/[A] at different, constant concentrations of the alternative substrate). The common-product approach, although very useful, will often produce nonlinearity in the double-reciprocal plots4,6,9. Huang has suggested that a constant-ratio approach, in which the substrate and the alternative substrate are varied in a constant ratio, can further assist an investigator in distinguishing between mechanisms6-8.

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.

ENERGETICS. While isozymes are apt to have different energies of activation, even under the same assay conditions, an enzyme acting on different substrates can in some circumstances exhibit the same energy of activa-tion10,11. Yeast sucrase, for example, has an energy of activation of 46 kJ-mol 1 (or 11.0 kcal-mol-1) for both sucrose and raffinose11-13. The rate-determining step in the enzyme-catalyzed reaction may differ with alternative substrates, and this may be reflected in the observed energy of activation. Likewise, if the rate-determining step changes with protein modification, assay conditions, or through site-directed mutagenesis, Arrhenius plots should reflect those changes. An example is the myosin ATPase which exhibits biphasicity in the Arrhenius plot with ITP as a substrate, but a typical linear Arrhenius plot with ATP as the substrate. Levy, Sharon, and Kosh-land14 suggested that this may be the result of the 6-amino group on ATP interacting with some functional moiety on the protein, thereby producing an enzymesubstrate complex insensitive to temperature change. See Competitive Inhibitor; Abortive Complexes; Map ping Substrate Interactions Using Substrate Data; Membrane Transport; Energy of Activation; Q10; Arrhenius Equation; van't Hoff Relationship

1J. T. Wong & C. S. Hanes (1962) Can. J. Biochem. Physiol. 40, 763.

2J. T. F. Wong (1975) Kinetics of Enzyme Mechanisms, Academic Press, New York.

3V. Zewe, H. J. Fromm & R. Fabino (1964) J. Biol. Chem. 239, 1625. 4H. J. Fromm (1964) Biochim. Biophys. Acta 81, 413. 5F. B. Rudolph & H. J. Fromm (1971) Arch. Biochem. Biophys. 147, 515.

7C. Y. Huang & S. Kaufman J. Biol. Chem. 248, 4242.

8C. Y. Huang (1977) Arch. Biochem. Biophys. 184, 488.

10M. Dixon & E. C. Webb, Enzymes, 3rd ed., Academic Press, New

York (1979). "I. W. Sizer (1943) Adv. Enzymol. 3, 35. 12I. W. Sizer (1938) Enzymologia 4, 215. 13I. W. Sizer (1937) J. Cellular and Comp. Physiol. 10, 61. 14H. M. Levy, N. Sharon & D. E. Koshland, Jr. (1959) Biochim. Bio-phys. Acta 33, 288.

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