T

Reaction Progress

Fig. 2-11. Alteration of the activation barrier for a chemical step in a multistep enzymatic mechanism. In a multistep mechanism, the several enzyme-substrate (ES) complexes designated Xj, X2, and so on may represent the products of substrate binding, conformational changes, and chemical transformations. In the plot, Xj may be the Michaelis complex, X2 a conformationally altered Michaelis complex, and X3 an enzyme product complex. The chemical reaction is the transformation of X2 to X3, a step that is normally not rate limiting because the barrier is lower than those of other steps. This reaction does not display a kinetic isotope effect. If an alternative substrate is used or a specifically mutated form of the enzyme is used, the barrier for the transformation of X2 to X3 may be raised (dashed line), making it the rate-limiting step. Then, normal kinetic isotope effects on the chemical step may be observed.

Reaction Progress

Fig. 2-11. Alteration of the activation barrier for a chemical step in a multistep enzymatic mechanism. In a multistep mechanism, the several enzyme-substrate (ES) complexes designated Xj, X2, and so on may represent the products of substrate binding, conformational changes, and chemical transformations. In the plot, Xj may be the Michaelis complex, X2 a conformationally altered Michaelis complex, and X3 an enzyme product complex. The chemical reaction is the transformation of X2 to X3, a step that is normally not rate limiting because the barrier is lower than those of other steps. This reaction does not display a kinetic isotope effect. If an alternative substrate is used or a specifically mutated form of the enzyme is used, the barrier for the transformation of X2 to X3 may be raised (dashed line), making it the rate-limiting step. Then, normal kinetic isotope effects on the chemical step may be observed.

of hydrogen (0.63 A), deuterium (0.45 A), and tritium (0.36 A) are near the distances these atoms traverse in crossing the transition-state barrier (=1 A), they are subject to tunneling through the barrier (Bahnson and Klinman, 1995; Klinman, 1991). Hydrogen tunneling is possible in any enzymatic reaction in which hydrogen is transferred. This is a quantum mechanical effect, as distinguished from the semiclassical vibrational effect in fig. 2-9. Tunneling imposes consequences on the kinetic isotope effects and can lead to large, nonclas-sical isotope effects. A narrow barrier as illustrated in fig. 2-12 can favor hydrogen tunneling.

Detailed analyses of hydrogen isotope effects in hydride transfer by alcohol dehydro-genase, hydrogen atom transfer by monoamine oxidase B, and proton transfer by serum amine oxidase uncovered hydrogen tunneling (Cha et al., 1989; Grant et al., 1989; Jonsson et al., 1994). Evidence of tunneling has also appeared in studies of adenosylcobalamin-dependent enzymes, which catalyze hydrogen atom transfer. Reactions with hydrogen tunneling constitute a broad range of reaction types involving hydride, hydrogen atom, and proton transfer.

Violations of the rule of the geometric mean in hydrogen transfer indicate hydrogen tunneling (Bahnson & Klinman, 1995; Klinman, 1991). According to this rule, the observed kinetic isotope effect is the product of primary and secondary effects in the same transition state (Bigeleisen, 1955). This means that the heavy isotopes operate independently on the energy of the transition state. A violation of this rule is evidence of hydrogen tunneling, and for this reason, the measurement of secondary isotope effects is important in studies of tunneling.

In the reaction of yeast alcohol dehydrogenase, hydride transfer displays a primary kinetic isotope effect, and a hydrogen in the same transition state but not transferred displays a secondary kinetic isotope effect. Such a hydrogen would be on in the nontrans-ferring position of the —CH2OH group or the C4(H) of NAD+. The first indication of hydrogen tunneling in these reactions appeared in the values of the secondary isotope effects, which were very large (1.22 to 1.35) compared with the equilibrium isotope effects of 0.9 to 1.04 (Cook et al., 1981; Klinman, 1991; Kurz and Frieden, 1980; Welsh et al., 1980).

Reaction progress

Fig. 2-12. Hydrogen tunneling through a narrow barrier. The dotted lines indicate the large free energy differences associated with protium (H), deuterium (D) and tritium (T) transfer from heteroatom A to heteroatomB.

Coupled motions of the transferred and nontransferred hydrogens in the transition state contributed to this effect but could not fully account for it without invoking hydrogen tunneling (Huskey and Schowen, 1983).

Two additional experimental tests for hydrogen tunneling can be applied (Bahnson & Klinman, 1995; Klinman, 1991). First, a breakdown of an alternative expression of the Swain-Schaad relationship, Tk = (Dk)L44, indicates tunneling. The exponent in the alternative relationship (kH/kT) = (kD/kT)3'26-3'34 lies in a narrow range, and a larger experimental value indicates hydrogen tunneling. Accurate values of kD/kT can be measured in a single reaction with tritium as a trace label by using deuterium as a trace label together with 14C as a remote label. The ratio of 3H/14C in the initial product is then directly related to kD/kT. Second, anomalous temperature effects on the rates of hydrogen and tritium transfer can indicate tunneling. Hydrogen and tritium transfer will display the same Arrhenius prefac-tor (AH) when there is no tunneling. The Arrhenius prefactor is obtained in plots of ln k against 1/T based on the equation ln kH = ln AH + £A(H)/RT. Different Arrhenius prefac-tors for H and T indicate hydrogen tunneling. The narrow temperature range over which enzymes can be studied hampers the application of this method. In all experiments to investigate hydrogen tunneling, the hydrogen transfer step must be rate limiting.

Hydrogen tunneling is most likely when hydrogen moves a short distance in traversing the transition state. A narrow barrier can bring about this condition (see fig. 2-12). Binding interactions in active sites of enzymes set up conditions for hydrogen tunneling. Because tunneling leads to very large kinetic isotope effects, the structure of a transition state for hydrogen transfer cannot be assigned based solely on the magnitude of the primary kinetic isotope effect. Carbon, with a de Broglie wavelength of 0.18 A, and other heavier atoms do not tunnel, and the primary and secondary kinetic isotope effects may be indicative of transition-state structure in accord with semiclassical models.

Transient-Phase Kinetics

Was this article helpful?

0 0

Post a comment