Quantitative description of absorption spectra of a pyridoxal phosphate-dependent enzyme using lognormal distribution curves

Metzler, Carol M.; Metzler, David E.

doi:10.1016/0003-2697(87)90580-x

Cited by 57 publications

(48 citation statements)

References 41 publications

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“…The high-molecular-weight species had absorbance maxima at 278, 331, and 416 nm, consistent with a PLP internal aldimine (45). Based on published molar absorption coefficients for PLP enzymes, 70% of the purified MJ0959 protein is bound to PLP (27).…”

Section: Maripaludismentioning

confidence: 99%

Biosynthesis of Phosphoserine in the Methanococcales

et al. 2007

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Methanococcus maripaludis and Methanocaldococcus jannaschii produce cysteine for protein synthesis using a tRNA-dependent pathway. These methanogens charge tRNA Cys with L-phosphoserine, which is also an intermediate in the predicted pathways for serine and cystathionine biosynthesis. To establish the mode of phosphoserine production in Methanococcales, cell extracts of M. maripaludis were shown to have phosphoglycerate dehydrogenase and phosphoserine aminotransferase activities. The heterologously expressed and purified phosphoglycerate dehydrogenase from M. maripaludis had enzymological properties similar to those of its bacterial homologs but was poorly inhibited by serine. While bacterial enzymes are inhibited by micromolar concentrations of serine bound to an allosteric site, the low sensitivity of the archaeal protein to serine is consistent with phosphoserine's position as a branch point in several pathways. A broad-specificity class V aspartate aminotransferase from M. jannaschii converted the phosphohydroxypyruvate product to phosphoserine. This enzyme catalyzed the transamination of aspartate, glutamate, phosphoserine, alanine, and cysteate. The M. maripaludis homolog complemented a serC mutation in the Escherichia coli phosphoserine aminotransferase. All methanogenic archaea apparently share this pathway, providing sufficient phosphoserine for the tRNA-dependent cysteine biosynthetic pathway.Many methanogenic archaea have a remarkable tRNA-dependent pathway for cysteine biosynthesis. In these organisms, an unusual class II aminoacyl-tRNA synthetase catalyzes the aminoacylation of tRNA Cys with L-phosphoserine (Sep) (38). This Sep-tRNA Cys is then sulfurylated to produce Cys-tRNA Cys for protein biosynthesis. This tRNA-dependent pathway for cysteine biosynthesis conserves the energy of a phosphoester bond compared to the canonical pathway, where phosphoserine is hydrolyzed to produce serine that is acetylated and then converted to cysteine by a sulfhydrolase. Methanocaldococcus jannaschii also uses phosphoserine to produce cystathionine (50). This paper addresses the biosynthesis of phosphoserine in Methanococcus maripaludis and M. jannaschii to decipher the relationship between cysteine and serine biosynthesis in the euryarchaea. Three pathways for the biosynthesis of serine are the phosphorylated pathway, which produces a phosphoserine intermediate (16, 18), the nonphosphorylated pathway (36), and the serine pathway for incorporating formaldehyde by the reverse activity of serine hydroxymethyltransferase (34). Most microorganisms use the phosphorylated pathway, which requires three dedicated enzymes, namely, phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase, and phosphoserine phosphatase. Many eukaryotes and some bacteria employ the nonphosphorylated pathway, which requires phosphoglycerate kinase, glycerate dehydrogenase, and serinepyruvate aminotransferase. The serine pathway for C 1 assimilation is commonly found in methanotrophic and methylotrophic bacteria.Isotopic labeling...

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Section: Maripaludismentioning

confidence: 99%

Biosynthesis of Phosphoserine in the Methanococcales

et al. 2007

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“…Thus, the first process is not PMP formation from a fast form of the enzyme. The imine nitrogen-protonated ketoenamine form of PLP absorbs at 420 nm, while the phenolic oxygen-protonated enolimine absorbs at 330 nm (20); thus, the observed spectral changes are consistent with a shift in the ratio of PLP tautomers. The second process observed is PMP formation where k 2 ) k decarboxylation .…”

Section: X-ray Crystallographic Strctures Of Q52a-plp and Wt Dgd-pmpmentioning

confidence: 57%

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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“…According to the results obtained for some Schiff bases of PLP with various amines and amino acids in media of different polarity (Metzler et al, 1980;Mitra and Metzler, 1988;Morozov et al, 1988;Vaizquez et al, 1991a,b) and investigations on PLPdependent enzymes (Kallen et al, 1985;Metzler and Metzler, 1987;Metzler et al, 1988;Miura et al, 1989), the three bands correspond to as many different tautomers of the coenzyme. Such tautomers (Scheme 1) are the zwitterionic form (Ba), the ion dipole form (Bb) and the enolimine (Be).…”

Section: Introductionmentioning

confidence: 98%

“…In dealing with these effects it may be of interest to determine the fractions of the chemical species that yield the different absorption bands. In this respect, deconvoluting enzyme spectra into log-normal distribution curves for PLP-dependent enzymes such as aspartate aminotransferase and glutamate decarboxylase (Kallen et al, 1985;Metzler and Metzler, 1987;Metzler et al, 1988;Miura et al, 1989) was found to be particularly useful for investigating potential reaction intermediates and mechanisms.…”

Section: Introductionmentioning

confidence: 99%

“…In the other PLP-dependent enzymes (Kallen et al, 1985;Metzler and Metzler, 1987;Metzler et al, 1988;Miura et al, 1989), where the cleavage of the internal imine bond and hence the protonation state of the pyridine nitrogen as an electron sink play a major role in the catalytic process, uptake of the substrates at the catalytic site substantially alters the distribution of the coenzyme tautomers. The little difference between the distribution of these forms in the activated (R) and inactive state (T) in glycogen phosphorylase must be a result of little, or even no, involvement of the aromatic ring of the coenzyme in the catalytic process, so the differences actually found probably suggest that the aromatic ring of PLP plays a merely structural role (Chang et al, 1987).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Quantitative description of the absorption spectra of the coenzyme in glycogen phosphorylases based on log-normal distribution curves

Donoso

Muñoz

Blanco

1993

Biochemical Journal

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The absorption spectra of the coenzyme [pyridoxal 5′-phosphate (PLP)] in glycogen phosphorylase a (GPha), glycogen phosphorylase b (GPhb) and of the latter bound to various effectors and substrates were analysed on the basis of log-normal distribution curves. The results obtained showed that the ionization state of the PLP and GPha environment differs from that of GPhb. This divergence was interpreted in terms of tautomeric equilibria between some forms of the Schiff base of PLP and enzymic Lys-679. The ionic forms are slightly more predominant in GPha than they are in GPhb, so ionic and/or hydrogen-bonding interactions between the aromatic ring of PLP and GPha must be stronger than with GPhb. This confirms the purely structural role of the aromatic ring of the coenzyme. Binding of GPhb to AMP and Mg2+ results in the coenzyme adopting a similar state as in GPha. On the other hand, binding to IMP gives rise to no detectable changes in the tautomeric equilibrium of the coenzyme.

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Quantitative description of absorption spectra of a pyridoxal phosphate-dependent enzyme using lognormal distribution curves

Cited by 57 publications

References 41 publications

Biosynthesis of Phosphoserine in the Methanococcales

Biosynthesis of Phosphoserine in the Methanococcales

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

Quantitative description of the absorption spectra of the coenzyme in glycogen phosphorylases based on log-normal distribution curves

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