Role of the pyridine nitrogen in pyridoxal 5′-phosphate catalysis: Activity of three classes of PLP enzymes reconstituted with deazapyridoxal 5′-phosphate

Wait R. Griswold, Michael D. Toney

Research output: Contribution to journalArticle

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Abstract

Pyridoxal 5′-phosphate (PLP; vitamin B 6)-catalyzed reactions have been well studied, both on enzymes and in solution, due to the variety of important reactions this cofactor catalyzes in nitrogen metabolism. Three functional groups are central to PLP catalysis: the C4′ aldehyde, the O3′ phenol, and the N1 pyridine nitrogen. In the literature, the pyridine nitrogen has traditionally been assumed to be protonated in enzyme active sites, with the protonated pyridine ring providing resonance stabilization of carbanionic intermediates. This assumption is certainly correct for some PLP enzymes, but the structures of other active sites are incompatible with protonation of N1, and, consequently, these enzymes are expected to use PLP in the N1-unprotonated form. For example, aspartate aminotransferase protonates the pyridine nitrogen for catalysis of transamination, while both alanine racemase and O-acetylserine sulfhydrylase are expected to maintain N1 in the unprotonated, formally neutral state for catalysis of racemization and β-elimination. Herein, kinetic results for these three enzymes reconstituted with 1-deazapyridoxal 5′-phosphate, an isosteric analogue of PLP lacking the pyridine nitrogen, are compared to those for the PLP enzyme forms. They demonstrate that the pyridine nitrogen is vital to the 1,3-prototropic shift central to transamination, but not to reactions catalyzed by alanine racemase or O-acetylserine sulfhydrylase. Not all PLP enzymes require the electrophilicity of a protonated pyridine ring to enable formation of carbanionic intermediates. It is proposed that modulation of cofactor electrophilicity plays a central role in controlling reaction specificity in PLP enzymes.

Original languageEnglish (US)
Pages (from-to)14823-14830
Number of pages8
JournalJournal of the American Chemical Society
Volume133
Issue number37
DOIs
StatePublished - Sep 21 2011

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Pyridoxal Phosphate
Catalysis
Pyridine
Catalyst activity
Phosphates
Nitrogen
Enzymes
Alanine Racemase
Cysteine Synthase
Catalytic Domain
Enzyme kinetics
Vitamin B 6
pyridine
Vitamins
Protonation
Aspartate Aminotransferases
Phenol
Aldehydes
Metabolism
Functional groups

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Role of the pyridine nitrogen in pyridoxal 5′-phosphate catalysis : Activity of three classes of PLP enzymes reconstituted with deazapyridoxal 5′-phosphate. / Griswold, Wait R.; Toney, Michael D.

In: Journal of the American Chemical Society, Vol. 133, No. 37, 21.09.2011, p. 14823-14830.

Research output: Contribution to journalArticle

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abstract = "Pyridoxal 5′-phosphate (PLP; vitamin B 6)-catalyzed reactions have been well studied, both on enzymes and in solution, due to the variety of important reactions this cofactor catalyzes in nitrogen metabolism. Three functional groups are central to PLP catalysis: the C4′ aldehyde, the O3′ phenol, and the N1 pyridine nitrogen. In the literature, the pyridine nitrogen has traditionally been assumed to be protonated in enzyme active sites, with the protonated pyridine ring providing resonance stabilization of carbanionic intermediates. This assumption is certainly correct for some PLP enzymes, but the structures of other active sites are incompatible with protonation of N1, and, consequently, these enzymes are expected to use PLP in the N1-unprotonated form. For example, aspartate aminotransferase protonates the pyridine nitrogen for catalysis of transamination, while both alanine racemase and O-acetylserine sulfhydrylase are expected to maintain N1 in the unprotonated, formally neutral state for catalysis of racemization and β-elimination. Herein, kinetic results for these three enzymes reconstituted with 1-deazapyridoxal 5′-phosphate, an isosteric analogue of PLP lacking the pyridine nitrogen, are compared to those for the PLP enzyme forms. They demonstrate that the pyridine nitrogen is vital to the 1,3-prototropic shift central to transamination, but not to reactions catalyzed by alanine racemase or O-acetylserine sulfhydrylase. Not all PLP enzymes require the electrophilicity of a protonated pyridine ring to enable formation of carbanionic intermediates. It is proposed that modulation of cofactor electrophilicity plays a central role in controlling reaction specificity in PLP enzymes.",
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