ARTICLE pubs.acs.org/JPCB
Multiphoton Manipulations of Enzymatic Photoactivity in Aspartate Aminotransferase Melissa P. Hill, Lucy H. Freer, Mai C. Vang, Elizabeth C. Carroll, and Delmar S. Larsen* Department of Chemistry, University of California—Davis, 1 Shields Avenue, Davis, California 95616, United States
bS Supporting Information ABSTRACT: The aspartate aminotransferase (AAT) enzyme utilizes the chromophoric pyridoxal 50 -phosphate (PLP) cofactor to facilitate the transamination of amino acids. Recently, we demonstrated that, upon exposure to blue light, PLP forms a reactive triplet state that rapidly (in microseconds) generates the high-energy quinonoid intermediate when bound to PLP-dependent enzymes [J. Am. Chem. Soc. 2010, 132 (47), 16953-16961]. This increases the net catalytic activity (kcat) of AAT, since formation of the quinonoid is partially rate limiting via the thermally activated enzymatic pathway. The magnitude of observed photoenhancement initially scales linearly with pump fluence; however when a critical threshold is exceeded, the photoactivity saturates and is even suppressed at greater excitation fluences. The photodynamic mechanisms associated with this suppression behavior are characterized with the use of ultrafast multipulse pump-dump-probe and pump-repump-probe transient absorption techniques in combination with complementary two-color, steady-state excitation assays. Via multistate kinetic modeling of the transient ultrafast data and the steady-state assay data, the nonmonotonic incident power dependence of the photoactivty in AAT is decomposed into contributions from high-intensity dumping of the excited singlet state and repumping of the excited triplet state with induces the repopulation of the ground state via rapid intersystem crossing in the higher-lying triplet electronic manifold.
1. INTRODUCTION Although many biological processes are initiated by the absorption of light, including photosynthesis, phototaxis, phototropism, gene expression, DNA repair, and stress responses,1 the vast majority of biological reactions do not require photoexcitation to operate. Nevertheless, many non-light-activated biological processes still use chromophoric cofactors that are capable of photoexcitation (e.g., hemes in myoblogin and hemoglobin2-4 and vitamin B6- and B12-dependent enzymes5). Consequently, light illumination may alter thermally activated function in such systems, potentially by manipulating cofactor-dependent kinetics or introducing new chemistry pathways. Previously, we demonstrated that the catalytic activity of aspartate aminotransferase (AAT), a vitamin B6-dependent enzyme, can be more than doubled upon continuous illumination of 440 nm light.6 Moreover, this light-enhanced enzymatic activity can be used to characterize thermally activated microscopic rate constants and evaluate microscopic models.7 Understanding the molecular mechanisms that couple photon absorption to enzymatic activity is important for developing light enhancement as a useful tool to explore enzymatic mechanisms. Approximately 4% of known enzymes use vitamin B6, also known as pyridoxal 50 -phosphate (PLP), to catalyze a variety of important biological reactions, including transamination, racemization, decarboxylation and many others.8 Aspartate aminotransferase is one such PLP-dependent enzyme, which facilitates the transamination of aspartate and R-ketoglutarate to generate r 2011 American Chemical Society
oxaloacetate and glutamate, respectively,8-13 and is a vital enzyme in numerous organisms required for amino acid synthesis as well as for replenishment of citric acid cycle intermediates essential for metabolism.14 AAT is central to nitrogen metabolism in all living systems and is a component of the malate/aspartate shuttle that provides reducing equivalents into eukaryotic mitochondria.8 PLP is bound to AAT’s active site as an internal Schiff base (SB) with a lysine residue (K258). AAT catalyzes the reversible reaction of L-aspartate with R-ketoglutarate to give oxaloacetate and Lglutamate via two independent half-reactions (eqs 1a and 1b).8-10 E-PLP þ L-Asp H E-PMP þ oxaloacetate
ð1aÞ
E-PMP þ R-ketoglutarate H E-PLP þ L-Glu
ð1bÞ
The reaction mechanism of AAT begins with PLP bound as a SB with K258 forming the “internal aldimine” (Scheme 1). Amino acid substrates (i.e., L-aspartate or L-glutamate) bind and form the covalent “external aldimine” (EA) SB by displacing K258 from the internal aldimine. It is generally thought that the next step involves the deprotonation of the L-Asp external aldimine at CR to form the carbanionic quinonoid intermediate, which protonates at C40 to generate an oxalacetate ketimine Received: December 30, 2010 Revised: February 15, 2011 Published: March 25, 2011 4474
dx.doi.org/10.1021/jp112400g | J. Phys. Chem. B 2011, 115, 4474–4483
The Journal of Physical Chemistry B
ARTICLE
Scheme 1. Mechanism of the Thermally Activated Transamination Half-Reaction from the EA Intermediate onward for AAT
Figure 1. Overlap of SB triplet spectrum (black) with (A) ground-state SB absorbance, fluorescence and laser emissions and (B) ground-state AAT external aldimine absorbance with LED emissions as labeled in the legends.
intermediate. This ketimine then hydrolyzes to give the pyridoxamine 50 -phosphate (PMP) enzyme form and free oxalacetate. The separate half-reaction converts E-PMP back to E-PLP using R-ketoglutarate as the second substrate (eq 1b).15,16 PLP typically exhibits absorption bands at ∼430, ∼360, and ∼330 nm with extinction coefficients of ∼5000 M-1 cm-1 (Figure 1); hence the blue edge of the solar spectrum is resonant with the PLP absorption. The previously proposed mechanism underlying the observed photoactivity in AAT involves the formation of an electronically excited triplet state formed via high yield intersystem crossing dynamics from a photoexcited singlet state (Figure 2).17 In the thermally activated pathway, EA loses Hþ from CR to form a resonantly stabilized, carbanionic “quinonoid” intermediate (Scheme 1). In the photoactivated pathway, the CR-H pKa in the triplet state is reduced from between 11 and 19 units,6 which promotes a faster deprotonation (∼μs) and greater population of quinonoid intermediates than in the thermally activated pathway (milliseconds). Since this step is partially rate limiting for overall enzymatic activity of AAT, the faster formation of the quinonoid intermediate results in faster
Figure 2. (A) Power-dependent kcat for wild-type AAT assay with simulated multistate saturation fit from previous proposed model using multiple turnover (high substrate concentration) conditions. (B) Excited-state reaction diagram with indicated transition wavelengths of constitute population involved in the first step of Scheme 1. Abbreviations: PLP-Asp SB, internal conversion time constant (τIC), intersystem crossing time constant (τISC), and excited-state proton transfer timeconstant (τESPT).
enzymatic activity. After quinonoid formation, the photoinitiated pathway continues to follow the same thermal pathway with an amino group transfer from L-aspartate (L-Asp) that converts PLP into PMP and L-Asp into oxaloacetate in the first half-reaction of a “ping-pong” kinetic mechanism. The second half-reaction converts PMP back to PLP and R-ketoglutarate to L-glutamate (eq 1b).15,16 The photochemistry of PLP-amino acid SBs in solution follows a similar reaction, whereby photoexcitation initiates a comparable transamination mechanism to form PMP.17 4475
dx.doi.org/10.1021/jp112400g |J. Phys. Chem. B 2011, 115, 4474–4483
The Journal of Physical Chemistry B The light-induced enhanced catalytic activity (referred to herein as photoactivity) of AAT exhibits a nonlinear and nonmonotonic dependence on light fluence (Figure 2A). Quantitative modeling of the fluence dependence of kcat is critical not only for quantifying the photophysics of PLP containing systems, but also for evaluating the role of light activation in chromophore-protein complexes. In the low to mid-intensity regime (
