Light enhanced enzyme catalysis and its implications for deciphering thermal enzyme mechanisms

The active form of vitamin B6, pyridoxal 5'-phosphate (PLP), is an important cofactor for numerous enzymes in amine and amino acid metabolism. This thesis combines various forms of time-resolved spectroscopies to investigate the light-activated dynamics of PLP-dependent enzymatic and non-enzymatic systems. The results from these studies have allowed greater insight into the thermally-activated mechanism of PLP-dependent enzymes as well as provided a foundation for the use of irradiated spectroscopy to interrogate thermally-activated enzymes. In Chapter 2, the first femtosecond transient absorption study of free PLP and two Schiff bases, PLP-valine and PLP-alpha-aminoisobutyric acid (AIB), in solution is presented. The construction and application of a single source femtosecond-millisecond broadband spectrometer is described in Chapter 3 where the utility of the system is demonstrated by measuring triplet state dynamics following photoexcitation of PLP. The photoactivation of the PLP-dependent enzyme aspartate aminotransferase (AAT) is described in Chapter 4 where characterization of this photoactivity results in a detailed mechanism for the light-enhancement. The mechanism includes excitation into an excited singlet state that decays into an excited triplet state where the pKa of the Calpha-H is lowered generating a vital reaction intermediate faster compared with the thermal mechanism thus enhancing the overall catalytic rate. This detailed mechanism leads to subsequent determination of microscopic rate constants for the thermally-activated system. Further interrogation into this photoactivation, including a high-power-dependence of the catalytic activity is the subject of Chapter 5, which concludes that suppression of the light-enhanced activity occurs at higher powers due to an overlap between the excited state triplet state and the excitation source. Definitive evidence for increased quinonoid intermediate concentration in the presence of light is provided in Chapter 6 with the use of a light-emitting diode-irradiated stopped flow instrument. In addition, several other PLP-dependent enzymes are assayed in the presence of light and also show catalytic enhancement. Light-enhancement of the deuterated substrate in AAT increases the rate of catalysis when compared to the protiated substrate resulting in an inverse kinetic isotope effect. The dominant reason for this observation is modification of the triplet lifetime upon deuteration; a topic that is thoroughly explored in Chapter 7.