Transient kinetics of the reaction catalysed by magnesium protoporphyrin IX methyltransferase

Abstract

Magnesium protoporphyrin IX methyltransferase (ChlM), an enzyme in the chlorophyll biosynthetic pathway, catalyses the transfer of a methyl group to magnesium protoporphyrin IX (MgP) to form magnesium protoporphyrin IX monomethyl ester (MgPME). S-Adenosyl-L-methionine is the other substrate, from which a methyl group is transferred to the propionate group on ring C of the porphyrin macrocycle. Stopped-flow techniques were used to characterize the binding of porphyrin substrate to ChlM from Synechocystis PCC6803 by monitoring tryptophan fluorescence quenching on a millisecond timescale. We concluded that a rapid binding step is preceded by a slower isomerization of the enzyme. Quenched-flow techniques have been employed to characterize subsequent partial reactions in the catalytic mechanism. A lag phase has been identified that has been attributed to the formation of an intermediate. Our results provide a greater understanding of this catalytic process which controls the relative concentrations of MgP and MgPME, both of which are implicated in signalling between the plastid and nucleus in plants.

Figure 1. Plots of observed rates of ChlM tryptophan fluorescence decay against MgD concentration

The data are fitted to a single hyperbolic decay, and the fitted parameters correspond to events in Scheme 1. The curve levels off at k_obs=3.09 s⁻¹, which corresponds to k₁. The fitted equilibrium constant is 3.36 μM, which corresponds to K₂. The y-axis intercept, corresponding to k₁+k₋₁·k₋₁, is approx. 600 s⁻¹.

Scheme 1. Proposed isomerization step that precedes MgP binding

Figure 2. Steady-state methyltransferase assay using 0.5 μM ChlM, 500 μM SAM and 100 μM MgD

The four chromatograms correspond to quenched aliquots taken at 2, 4, 6 and 8 min. The pigments were bound to a C-18 reversed-phase column with a 2 ml bed volume. A gradient of acetonitrile from 0–100% over 15 ml was used to elute substrate and product at different points. The elution of porphyrins was detected using a fluorescence detector with excitation and emission wavelengths of 400 nm and 580 nm respectively. Peaks at 10 min and 12.3 min correspond to MgD and MgDME respectively. The peaks at 12.7 min correspond to a putative intermediate.

Figure 3. Demonstration of an intermediate in the reaction mechanism using a combination of quenched-flow techniques and HPLC reversed-phase chromatography

(A) HPLC chromatograms demonstrating the pre-steady-state turnover of 0.5 μM ChlM with 1 mM SAM and 30 μM MgD. The elution of porphyrins was detected using a fluorescence detector with excitation and emission wavelengths of 400 nm and 580 nm respectively. This sample of chromatograms illustrates the evolution/depletion of intermediate and evolution of MgDME. (B) Evolution and depletion of the intermediate that is eluted at 12.7 min during HPLC analysis. Fluorescence excitation and emission wavelengths were 400 nm and 580 nm respectively. These data have been fitted to a double exponential. k₁=11.9±0.5 s⁻¹, k₂=11.8±0.5 s⁻¹. (C) Evolution of MgDME calculated from the peaks at 12.3 min on the HPLC chromatograms. MgDME concentrations have been fitted to a double exponential with a linear phase, using the rate constants from (B). The steady-state rate is 0.41±0.04 μM·min⁻¹.

Scheme 2. Proposed reaction mechanism of ChlM

The scheme depicts a random ternary mechanism, whereby both substrates may bind in either order. K_m^SAM=38 μM, K_d^MgD=2.37 μM, k_cat/K_m^SAM=1500 M⁻¹·s⁻¹, K_i≈K_i′ [21]. k₁=3.09 s⁻¹, k₋₁≈600 s⁻¹, K₂=3.36 μM. E, enzyme; E′, enzyme with altered conformation; Int, putative intermediate.