Rapid kinetic and isotopic studies on dialkylglycine decarboxylase.
The two half-reactions of the pyridoxal 5'-phosphate (PLP)-dependent enzyme dialkylglycine decarboxylase (DGD) were studied individually by multiwavelength stopped-flow spectroscopy. Biphasic behavior was found for the reactions of DGD-PLP, consistent with two coexisting conformations observed in steady-state kinetics [Zhou, X., and Toney, M. D. (1998) Biochemistry 37, 5761--5769]. The half-reaction kinetic parameters depend on alkali metal ion size in a manner similar to that observed for steady-state kinetic parameters. The fast phase maximal rate constant for the 2-aminoisobutyrate (AIB) decarboxylation half-reaction with the potassium form of DGD-PLP is 25 s(-1), while that for the transamination half-reaction between DGD-PMP and pyruvate is 75 s(-1). The maximal rate constant for the transamination half-reaction of the potassium form of DGD-PLP with L-alanine is 24 s(-1). The spectral data indicate that external aldimine formation with either AIB or L-alanine and DGD-PLP is a rapid equilibrium process, as is ketimine formation from DGD-PMP and pyruvate. Absorption ascribable to the quinonoid intermediate is not observed in the AIB decarboxylation half-reaction, but is observed in the dead-time of the stopped-flow in the L-alanine transamination half-reaction. The [1-(13)C]AIB kinetic isotope effect (KIE) on k(cat) for the steady-state reaction is 1.043 +/- 0.003, while a value of 1.042 +/- 0.009 was measured for the AIB half-reaction. The secondary KIE measured for the AIB decarboxylation half-reaction with [C4'-(2)H]PLP is 0.92 +/- 0.02. The primary [2-(2)H]-L-alanine KIE on the transamination half-reaction is unity. Small but significant solvent KIEs are observed on k(cat) and k(cat)/K(M) for both substrates, and the proton inventories are linear in each case. NMR measurements of C2--H washout vs product formation give ratios of 105 and 14 with L-alanine and isopropylamine as substrates, respectively. These results support a rate-limiting, concerted C alpha-decarboxylation/C4'-protonation mechanism for the AIB decarboxylation reaction, and rapid equilibrium quinonoid formation followed by rate-limiting protonation to the ketimine intermediate for the L-alanine transamination half-reaction. Energy profiles for the two half-reactions are constructed.
Zhou
X
X
Department of Chemistry, University of California--Davis, One Shields Avenue, Davis, California 95161, USA.
Jin
X
X
Medhekar
R
R
Chen
X
X
Dieckmann
T
T
Toney
M D
MD
D000409:Alanine / Q000737:chemistry; D000621:Aminoisobutyric Acids / Q000737:chemistry; D002247:Carbon Isotopes; D002262:Carboxy-Lyases / Q000737:chemistry*; D003653:Decarboxylation; D003903:Deuterium; D004789:Enzyme Activation; D007527:Isoenzymes / Q000737:chemistry; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D011522:Protons; D011732:Pyridoxal Phosphate / Q000737:chemistry; D019289:Pyruvic Acid / Q000737:chemistry; D000637:Transaminases / Q000737:chemistry
D000621:Aminoisobutyric Acids; D002247:Carbon Isotopes; D007527:Isoenzymes; D011522:Protons; C100049:2-aminoisobutyric acid; D011732:Pyridoxal Phosphate; D019289:Pyruvic Acid; D003903:Deuterium; D000637:Transaminases; D002262:Carboxy-Lyases; C063519:2,2-dialkylglycine decarboxylase; D000409:Alanine