Interaction of the apoenzyme of L‐glutamate decarboxylase with pyridoxal phosphate analogues

Mechanik, M.L.; Torchinsky, Yu.M.; Florentiev, V. L.; Karpeisky, M.Ya.

doi:10.1016/0014-5793(71)80229-6

Cited by 8 publications

(8 citation statements)

References 5 publications

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“…Reporting on natural PLP binding events requires a functional, minimally-modified synthetic cofactor probe capable of labeling enzymes covalently. Previous investigations found that modifications at the 2-position of PLP, including either removal or limited expansion of the methyl group, could be tolerated by different PLP-DEs 15–17. Following the example of activity-based protein profiling,18 we designed PL-probes containing a small alkyne tag either directly attached to the pyridine ring ( PL1 ) or with an ethylene spacer ( PL2 ), and a 2’-azide analogue ( PL3 ) to account for chemical preferences within protein binding sites (Fig.…”

Section: Resultsmentioning

confidence: 99%

Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics

et al. 2018

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Pyridoxal phosphate (PLP) is an enzyme cofactor required for the chemical transformation of biological amines in numerous essential cellular processes. PLP-dependent enzymes (PLP-DEs) are ubiquitous and evolutionarily diverse, making their classification based on sequence homology challenging. Here we present a chemical proteomic method for reporting on PLP-DEs using functionalized cofactor probes. We synthesized pyridoxal (PL)-analogues modified at the 2’-position which are taken up by cells and metabolized in situ. These PL-analogues are phosphorylated to functional cofactor surrogates by cellular PL kinases and bind to PLP-DEs via an aldimine bond which can be rendered irreversible by NaBH 4 reduction. Conjugation to a reporter tag enables the subsequent identification of PLP-DEs using quantitative, label-free mass spectrometry. Using these probes we accessed a significant portion of the Staphylococcus aureus PLP-DE proteome (73%) and annotate uncharacterized proteins as novel PLP-DEs. We also show that this approach can be used to study structural tolerance within PLP-DE active sites and to screen for off-targets of the PLP-DE inhibitor D-cycloserine.

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Section: Resultsmentioning

confidence: 99%

Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics

et al. 2018

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“…As described in the introduction, the binding of pyridoxal-P to glutamate apodecarboxylase arises from separate contributions of the Schiff base linkage, the pyridine nitrogen, the aromatic ring, the 3-hydroxyl group, and the phosphate side chain. Based on the fact that 2-norpyridoxal-P closely approximates the normal coenzyme (Mekhanik et al, 1971), we assume that the 2-methyl group makes a negligible contribution to the binding. In addition, we assume that contributions of the various functional groups are additive.…”

Section: Resultsmentioning

confidence: 99%

“…The role of the phosphate ester group in binding is perhaps the most complex and the least understood. Coenzyme analogues lacking an anionic group at the 5 position bind at least 106-fold more poorly than the natural coenzyme (O'Leary, 1969;Fonda, 1971;Mekhanik et al, 1971Mekhanik et al, , 1972. Simple phosphates bind to the phosphate binding site, and this binding has been suggested to be the first step in the formation of the protein-pyridoxal-P complex (O'Leary & Malik, 1972).…”

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Coenzyme binding site of glutamate decarboxylase

O’Leary¹,

Koontz²

1980

Biochemistry

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Dissociation constants have been measured for the binding of a variety of simple analogues of pyridoxal 5'-phosphate to apoglutamate decarboxylase. Compounds studied have a simple alkyl or aryl group and a negatively charged substituent (phosphate, phosphonate, phosphoramidate, sulfate, sulfonate, or carboxylate). Optimum binding to the phosphate binding site of the enzyme is achieved by compounds having a double negative charge and a tetrahedral geometry. Planar anions and monoanions bind considerably less well. These and previous data are used to to derive the magnitudes of the contributions of various coenzyme functional groups to the strength of the apoenzyme-coenzyme interaction.

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“…5 and 11). Alternatively, binding of coenzyme to apoenzyme through both phosphate oxygens might be necessary to produce a catalytically competent conformation in the holoenzyme (1,4). Additional observations that shed light on these possibilities would be useful.…”

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confidence: 99%

“…This topic has been the subject of a recent review (1), and of several more recent publications (2)(3)(4)(5). For every apoenzyme so far studied, analogues of pyridoxal-P with an uncharged or a singly charged anionic group replacing the 5'-phosphate group have been either inactive or have shown only negligible activity as substitutes for pyridoxal-P. A particularly interesting analogue in this connection is pyridoxal 5'-sulfate (6), which has a single strongly acidic ionizable hydrogen on the esterified sulfate group, but is sterically closely similar to pyridoxal 5'-phosphate.…”

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Coenzymatic Activity of Pyridoxal 5′-Sulfate and Related Analogues of Pyridoxal 5′-Phosphate

Groman

Huang

Watanabe

et al. 1972

Proc. Natl. Acad. Sci. U.S.A.

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The effect of changes in the substituent at the 5'-position of pyridoxal 5'-phosphate on the coenzymatic activity of six analogues of this coenzyme was determined for three bacterial enzymes. Pyridoxal 5'-sulfate showed substantial coenzymatic activity for arginine decarboxylase and tryptophanase, but not for D-serine dehydratase; a5-pyridoxalmethylphosphonate showed substantial activity for D-serine dehydratase, but not for the other two enzymes. These results demonstrate that neither the dianionic phosphate group nor the ester oxygen of pyridoxal 5'-phosphate is a general requirement for coenzymatic activity. Minor variations in orientation and charge of the group at position 5 exert major effects on the capacity for complex formation between analogue and apoenzyme, and on the catalytic efficiency of the resulting complex, but have comparatively little effect on the substrate affinity of the analogue-apoenzyme complexes. These effects show no general pattern from enzyme to enzyme, and do not correlate with the affinity of analogue for apoenzyme under the conditions tested.

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Interaction of the apoenzyme of L‐glutamate decarboxylase with pyridoxal phosphate analogues

Cited by 8 publications

References 5 publications

Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics

Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics

Coenzyme binding site of glutamate decarboxylase

Coenzymatic Activity of Pyridoxal 5′-Sulfate and Related Analogues of Pyridoxal 5′-Phosphate

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