Reactions of Alternate Substrates Demonstrate Stereoelectronic Control of Reactivity in Dialkylglycine Decarboxylase

Sun, Shaoxian; Zabinski, Roger F.; Toney, Michael D.

doi:10.1021/bi972055s

Cited by 33 publications

(66 citation statements)

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“…The roles of several catalytically important pK a s, including one responsible for the interconversion of the two coexisting conformations, have been identified in an extensive pH study (7). Additionally, steady-state and pre-steady-state kinetic analyses of the DGD reactions with alternate substrates have shown that stereoelectronic effects play a role in determining decarboxylation vs transamination reactivity (8,9).The first step of all PLP-dependent enzymes is formation of an external aldimine intermediate via a transimination reaction, displacing the active site lysine from its interaction with PLP (Scheme 1). In a classical stepwise decarboxylation mechanism, loss of the AIB R-carboxylate group as CO 2 from the external aldimine intermediate leads to the quinonoid intermediate, which, on protonation at C4′, gives the ketimine intermediate.…”

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Rapid Kinetic and Isotopic Studies on Dialkylglycine Decarboxylase

et al. 2001

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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 halfreaction kinetic parameters depend on alkali metal ion size in a manner similar to that observed for steadystate 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 halfreaction with [C4′-2 H]PLP is 0.92 ( 0.02. The primary [2-2 H]-L-alanine KIE on the transamination halfreaction 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 CR-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.Dialkylglycine decarboxylase (DGD) 1 is an intriguing PLP-dependent enzyme because it catalyzes both transamination and decarboxylation reactions in its normal catalytic cycle (Scheme 1). The enzyme was isolated from a soil bacterium (1) and transfers the fixed nitrogen from small 2,2-dialkylglycines, which are common in peptide antibiotics (2), onto pyruvate to give L-alanine. The decarboxylation half-reaction is highly specific for oxidative decarboxylation. Thus, the issue of reaction specificity has two main facets with this enzyme: control of decarboxylation vs transamination, and control of oxidative vs nonoxidative decarboxylation. The latter is addressed in this work.Previous work has shown that DGD activity is dependent on alkali metal ions; potassium activates it w...

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Rapid Kinetic and Isotopic Studies on Dialkylglycine Decarboxylase

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“…sites (11). The A subsite (Figure 1) can bind a carboxylate in a position that is stereoelectronically activated for decarboxylation.…”

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“…The rate of decarboxylation in Q52E (k decarboxylation ) 6 × 10 -4 s -1 ) can be used as an estimate of the rate of decarboxylation from the nonactivated position by assuming that the Q52E mutation precludes carboxylate binding at the A subsite. The rate of decarboxylation from the activated A subsite can be estimated using the WT value for the AIB decarboxylation half-reaction and previous studies showing that approximately one-fifth of the substrate binds with the carboxylate in the A subsite (11), allowing the calculation of the rate of decarboxylation from the A subsite (k activated ) 125 s -1 ).…”

Section: Formation Of the Michaelis Complex And Formation Of The Extementioning

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“…A functional active site model based on the unusual reaction specificity of DGD and stereoelectronic control of reaction specificity has been proposed (14) and validated (11). In this model, there are three subsites, the A subsite, near Q52 and K272, the stereoelectronically activated position in which all bond breaking and making occur; the B subsite, near R406 and S215, which can bind a carboxylate; and the C subsite, near M141 ( Figure 1).…”

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“…A careful search of the literature shows no published examples of a PLP dependent enzyme catalyzing the cleavage of more than one bond to C R with its natural substrate, except DGD which catalyzes both transamination and decarboxylation with L-alanine (11). The found value of 7.3 kcal/mol corresponds to one side reaction every one in 10 6 turnovers, which is within the detection limits of studies of ornithine decarboxylase (30) and aspartate aminotransferase (31,32) that find no evidence of side reactions which differ in the bond to C R that is broken.…”

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Role of Q52 in Catalysis of Decarboxylation and Transamination in Dialkylglycine Decarboxylase

et al. 2005

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Dialkylglycine decarboxylase (DGD) is a pyridoxal phosphate dependent enzyme that catalyzes both decarboxylation and transamination in its normal catalytic cycle. DGD uses stereoelectronic effects to control its unusual reaction specificity. X-ray crystallographic structures of DGD suggest that Q52 is important in maintaining the substrate carboxylate in a stereoelectronically activated position. Here, the X-ray structures of the Q52A mutant and the wild type (WT) DGD-PMP enzymes are presented, as is the analysis of steady-state and half-reaction kinetics of three Q52 mutants (Q52A, Q52I, and Q52E). As expected if stereoelectronic effects are important to catalysis, the steady-state rate of decarboxylation for all three mutants has decreased significantly compared to that of WT. Q52A exhibits an ∼85-fold decrease in k cat relative to that of WT. The rate of the decarboxylation half-reaction decreases ∼10 5 -fold in Q52I and ∼10 4 -fold in Q52E compared to that of WT. Transamination half-reaction kinetics show that Q52A and Q52I have greatly reduced rates compared to that of WT and are seriously impaired in pyridoxamine phosphate (PMP) binding, with K PMP at least 50-100-fold greater than that of WT. The larger effect on the rate of L-alanine transamination than of pyruvate transamination in these mutants suggests that the rate decrease is the result of selective destabilization of the PMP form of the enzyme in these mutants. Q52E exhibits near-WT rates for transamination of both pyruvate and L-alanine. Substrate binding has been greatly weakened in Q52E with apparent dissociation constants at least 100-fold greater than that of WT. The rate of decarboxylation in Q52E allows the energetic contribution of stereoelectronic effects, ∆G stereoelectronic , to be estimated to be -7.3 kcal/mol for DGD.Pyridoxal phosphate (PLP) 1 dependent enzymes constitute a large and well-characterized group of enzymes, catalyzing many different types of chemistry at the R-, -, and γ-carbons of amine and amino acid substrates. Despite the diversity of chemistry catalyzed by PLP dependent enzymes as a group, individual enzymes exhibit remarkably high reaction specificities. In contrast, model studies show that PLP alone is capable of nonenzymatically catalyzing multiple reaction types with a given substrate. For example, reaction of PLP with serine results in transamination, -elimination, and retroaldol cleavage (1). As an explanation for the high enzymatic versus nonenzymatic specificity, it was proposed by Dunathan (2) that PLP dependent enzymes use stereoelectronic effects to control reaction specificity. He proposed that the scissile bond is aligned parallel to the p orbitals of the extended π system of the aldimine, providing maximal orbital overlap and resonance stabilization in the transition state, therefore accelerating the rate of bond cleavage for the activated bond compared to the other bonds to C R .Stereoelectronic effects have been investigated in simple organic systems both experimentally and computationally (...

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PLP‐Dependent Enzymes, Chemistry of

Toney

2008

Wiley Encyclopedia of Chemical Biology

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Pyridoxal phosphate‐dependent enzymes catalyze a wide variety of reaction types on amines and amino acids, generally by stabilizing carbanionic intermediates, which makes them very common in both primary and secondary metabolism. A fundamental mechanism by which reaction specificity is enforced is stereoelectronic effects; the bond to Cα that gets broken in the universal external aldimine intermediate is that which is aligned parallel to the p orbitals of the conjugated, electron‐deficient π system. When a bond to Cα has been broken, enzymes must also carefully control the fate of the resulting carbanionic intermediate. Structurally, all known pyridoxal 5′‐phosphate (PLP)‐dependent enzymes are separated into five groups. The largest of these, Type 1, is the aminotransferase group, of which aspartate aminotransferase is the prototype. The prototypes for the other groups are as follows: Type 2, tryptophan synthetase; Type 3, alanine racemase; Type 4, D‐amino acid aminotransferase; and Type 5, glycogen phosphorylase.

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Reactions of Alternate Substrates Demonstrate Stereoelectronic Control of Reactivity in Dialkylglycine Decarboxylase

Cited by 33 publications

References 32 publications

Rapid Kinetic and Isotopic Studies on Dialkylglycine Decarboxylase

Rapid Kinetic and Isotopic Studies on Dialkylglycine Decarboxylase

Role of Q52 in Catalysis of Decarboxylation and Transamination in Dialkylglycine Decarboxylase

PLP‐Dependent Enzymes, Chemistry of

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