PLP Model Chemistry

In the initial spectroscopic observation of product radical 3 (Fig. 3), LAM was purified in an anaerobic chamber and used to prepare 

Additional samples were prepared and frozen at various times in the course of the approach of the overall reaction to equilibrium, which required several minutes. Each sample was assayed for L- -lysine, and the EPR spectra were integrated to evaluate spin concentrations. The integrated spins in the samples were found to decrease with time to an equilibrium value. Approach to constant integrated spin took place with exactly the same time constant as that characterizing approach to chemical equilibrium for the overall reaction. Equality in rates suggested that the radical was an intermediate in the overall reaction, in which its maximum concentration existed at the steady state in the forward direction. A corollary of this interpretation is that in the reverse direction, the transformation of L-P-lysine into L-lysine, the radical signal must be small in the initial steady state and approach the equilibrium value as the overall reaction approaches equilibrium. This has been confirmed.  

To determine whether PLP was actually associated with the lysine radical, [4 - H]PLP was synthesized and exchanged into the enzyme, and the [4 - H]PLP-enzyme was used to prepare a sample of the putative product radical 3. The EPR spectrum of the sample containing [4 - H]PLP proved to be identical with that of a matched sample containing PLP. The two samples were submitted to electron spin echo envelope modulation spectroscopy (ESEEM). The ESEEM spectra revealed a signal corresponding to the Larmor frequency for deuterium in the sample containing [4 - H]PLP (Fig. 5) and no signal in the PLP sample. This meant that the deuterium in [4 - H]PLP must be 

Identification of radical 3 as a species that is present in the steady-state phase of the reaction does not prove that it is an intermediate—it could be a species that is peripheral to the real reaction mechanism. Proof that a species is an intermediate requires a demonstration that it is kinetically competent to participate in the mechanism. In the case of a metastable radical, the usual procedure is to conduct transient kinetic studies using a rapid mixing apparatus equipped to quench samples by spraying them into liquid isopentane. The frozen aqueous samples (snows) from the timed cold quenches are then packed into EPR tubes and analyzed spectroscopically. Simple mixing of enzyme with SAM and lysine followed by freeze-quenching on the millisecond time scale does not work because the activation by SAM takes about 5 s. However, a preliminary mix of enzyme with SAM and [2- C]lysine, aging of the solution for 5 s within the apparatus. 

---