Mass-dependent bond vibrational dynamics influence catalysis by HIV-1 protease

Abstract

Protein motions that occur on the microsecond to millisecond time scale have been linked to enzymatic rates observed for catalytic turnovers, but not to transition-state barrier crossing. It has been hypothesized that enzyme motions on the femtosecond time scale of bond vibrations play a role in transition state formation. Here, we perturb femtosecond motion by substituting all nonexchangeable carbon, nitrogen, and hydrogen atoms with (13)C, (15)N, and (2)H and observe the catalytic effects in HIV-1 protease. According to the Born-Oppenheimer approximation, isotopic substitution alters vibrational frequency with unchanged electrostatic properties. With the use of a fluorescent peptide to report on multiple steps in the reaction, we observe significantly reduced rates in the heavy enzyme relative to the light enzyme. A possible interpretation of our results is that there exists a dynamic link between mass-dependent bond vibrations of the enzyme and events in the reaction coordinate.

Figure 1

Circular dichroism (CD) spectral analysis. Spectra obtained from 10 µM light (left) and heavy (right) HIV-1 protease in 50 mM MES-Tris pH 6.0 and 1.25 M NaCl are shown. Each trace represents an average of five scans and each graph shows traces from three independent samples. An overlay of the heavy and light spectra is shown as Supplementary Information, Figure S2.

Figure 2

Saturation kinetics of light, ¹⁵N-labeled and heavy HIV-1 protease. Steady state kinetic rates of product formation are plotted as a function of fluorescent substrate concentration. Catalysts are 10 nM light (blue), ¹⁵N (pink), and heavy (black) HIV-1 protease. Curves were plotted as a function of initial rate (v_i) of product formation and substrate concentration and were fit to the equation v_i = k_cat[E][A]/(K_m + [A]).

Figure 3

Pre-steady-state burst kinetics. Substrate (100 µM) was mixed with either 10 µM light (blue) or heavy enzyme (black) and the reaction was monitored at 280 nm excitation with a >400 nm emission filter. Curves were fit to Equation 1 and the kinetic constants are listed in Table 1. Each dashed line represents the slope of the steady-state phase extrapolated to the y-axis, which is proportional to the concentration of the EX complex for the corresponding enzyme. The inset window shows first 30 ms of the reaction and illustrates the magnitude of the difference observed in the burst rate constants for the heavy and light enzyme.

Scheme 1

Kinetic mechanism of HIV-1 protease

Scheme 2

Simplified mechanism for pre-steady-state kinetic analysis