Light-enhanced catalysis by pyridoxal phosphate-dependent aspartate aminotransferase

The mechanisms of pyridoxal 5'-phosphate (PLP)-dependent enzymes require substrates to form covalent "external aldimine" intermediates, which absorb light strongly between 410 and 430 nm. Aspartate aminotransferase (AAT) is a prototypical PLP-dependent enzyme that catalyzes the reversible interconversion of aspartate and α-ketoglutarate with oxalacetate and glutamate. From kinetic isotope effects studies, it is known that deprotonation of the aspartate external aldimine C(α)-H bond to give a carbanionic quinonoid intermediate is partially rate limiting in the thermal AAT reaction. We show that excitation of the 430-nm external aldimine absorption band increases the steady-state catalytic activity of AAT, which is attributed to the photoenhancement of C(α)-H deprotonation on the basis of studies with Schiff bases in solution. Blue light (250 mW) illumination gives an observed 2.3-fold rate enhancement for WT AAT activity, a 530-fold enhancement for the inactive K258A mutant, and a 58600-fold enhancement for the PLP-Asp Schiff base in water. These different levels of enhancement correlate with the intrinsic reactivities of the C(α)-H bond in the different environments, with the less reactive Schiff bases exhibiting greater enhancement. Time-resolved spectroscopy, ranging from femtoseconds to minutes, was used to investigate the nature of the photoactivation of C(α)-H bond cleavage in PLP-amino acid Schiff bases both in water and bound to AAT. Unlike the thermal pathway, the photoactivation pathway involves a triplet state with a C(α)-H pK(a) that is estimated to be between 11 and 19 units lower than the ground state for the PLP-Val Schiff base in water.     

Adaptive Feedback Linearizing Control of Proper Orthogonal Decomposition Nonlinear Flow Models

This paper treats the question of feedback linearizing control oftwo-dimensional incompressible, unsteady wake flow. For definiteness,flow past a circular cylinder is considered, but the design approachpresented here is applicable to other flow control problems. Twofinite-dimensional lower-order models based on Proper OrthogonalDecomposition (POD) of dimension N with N actuators are considered.Models I and II are obtained using control function and penalty functionmethods, respectively. Control action can be achieved by a combinationof suction, injection, and synthetic jets. For the design ofcontrollers, it is assumed that the system matrices of the POD modelsare unknown. Nonlinear adaptive control systems for the two models arederived. For model I, nontrivial zero-error dynamics exists, which playa key role in the stability of the closed-loop system. But for model II,global adaptive trajectory control is achieved. In the closed-loopsystem, the mode amplitudes asymptotically follow the referencetrajectories. Simulation results for a 4-mode POD model obtained usingthe penalty function method are presented. These results show that inthe closed-loop system, unsteadiness in the mode amplitudes can besuppressed in spite of large uncertainties in the flow model.