Inactivation of yeast hexokinase by o-phthalaldehyde: evidence for the presence of a cysteine and a lysine at or near the active site

Puri, Rajinder N.; Bhatnagar, Deepak; Roskoski, Robert

doi:10.1016/0167-4838(88)90154-9

Cited by 29 publications

(19 citation statements)

References 43 publications

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“…In the catalytic site for glucose in hexokinase there are two glutamic residues (Glu-269 and Glu-302) and an aspartic (Asp-211) [20]. Furthermore, there is a cystein close to the active site that could take part in the catalysis [36]. Regarding the ATP binding site, an ATP binding pattern has been defined [20] in which there are residues of Gly, Ser, Thr and Asp strictly conserved between all the compared sequences studied.…”

Section: Resultsmentioning

confidence: 99%

Structural and functional implications of the hexokinase–nickel interaction

Romero

Olmo

Teijón

et al. 2005

Journal of Inorganic Biochemistry

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Section: Resultsmentioning

confidence: 99%

Structural and functional implications of the hexokinase–nickel interaction

Romero

Olmo

Teijón

et al. 2005

Journal of Inorganic Biochemistry

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“…We asked if o -phthaldialdehyde alone, without any kinase directed adenosine or aminopyrazole element, is capable of cross-linking cysteine containing peptide 2 to cellular proteins. o -Phthaldialdehyde has been used to intramolecularly label enzymes that possess proximal lysines and cysteines, such as creatine kinase28 and hexokinase,29 and thus is itself capable of mediating isoindole formation in a cellular context. We asked if o -phthaldialdehyde would efficiently carry out intermolecular cross-linking of peptide 2 to cellular proteins.…”

Section: Resultsmentioning

confidence: 99%

Tuning a Three-Component Reaction For Trapping Kinase Substrate Complexes

et al. 2008

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The upstream protein kinases responsible for thousands of phosphorylation events in the phosphoproteome remain to be discovered. We developed a three-component chemical reaction which converts the transient non-covalent substrate-kinase complex into a covalently cross-linked product by utilizing a dialdehyde based cross-linker, 1. Unfortunately the reaction of 1 with a lysine in the kinase active site and an engineered cysteine on the substrate to form an isoindole cross-linked product could not be performed in the presence of competing cellular proteinsdue to non-specific side reactions. In order to more selectively target the cross-linker to protein kinases in cell lysates we replaced the weak, kinase-binding adenosine moiety of 1 with a potent protein kinase inhibitor scaffold. In addition, we replaced the o-phthaldialdehyde moiety in 1 with a less reactive thiophene-2,3-dicarboxaldehyde moiety. The combination of these two structural modifications provides for cross-linking of a cysteine containing substrate to its corresponding kinase in the presence of competing cellular proteins.

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“…16 E T ¼ 2:985l em 2 1087:28: The E T value has been used by many investigators 18,19 to probe the polarity of the microenvironment of participating cysteine and lysine residues by comparing it with the values of a synthetic thioisoindole OPA adduct in different solvents. E T for the adducts has been reported to be 127 kJ mol 21 in hexane, 155 kJ mol 21 in dioxane and 267 kJ mol 21 in water.…”

Section: Enzyme Inactivation With O-phthalaldehydementioning

confidence: 99%

Chemical Modification of Specific Active Site Amino Acid Residues of Enterobacter aerogenes Glycerol Dehydrogenase

Pandey

Iyengar

2002

Journal of Enzyme Inhibition and Medicinal Chemistry

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Enterobacter aerogenes glycerol dehydrogenase (G1DH EC 1.1.1.6), a tetrameric NAD+ specific enzyme catalysing the interconversion of glycerol and dihydroxyacetone, was inactivated on reaction with pyridoxal 5-phosphate (PLP) and o-phthalaldehyde (OPA). Fluorescence spectra of PLP-modified, sodium borohydride-reduced G1DH indicated the specific modification of epsilon-amino groups of lysine residues. The extent of inhibition was concentration and time dependent. NAD+ and NADH provided complete protection against enzyme inactivation by PLP, indicating the reactive lysine is at or near the coenzyme binding site. Modification of G1DH by the bifunctional reagent OPA, which reacts specifically with proximal epsilon-NH2 group of lysines and -SH group of cysteines to form thioisoindole derivatives, inactivated the enzyme. Molecular weight determinations of the modified enzyme indicated the formation of intramolecular thioisoindole formation. Glycerol partially protected the enzyme against OPA inactivation, whereas NAD+ was ineffective. These results show that the lysine involved in the OPA reaction is different from the PLP-reactive lysine, which is at or near the coenzyme binding site. DTNB titration showed the presence of only a single cysteine residue per monomer of G1DH. This could be participating with a proximal lysine residue to form a thioisoindole derivative observed as a result of OPA modification.

show abstract

Inactivation of yeast hexokinase by o-phthalaldehyde: evidence for the presence of a cysteine and a lysine at or near the active site

Cited by 29 publications

References 43 publications

Structural and functional implications of the hexokinase–nickel interaction

Structural and functional implications of the hexokinase–nickel interaction

Tuning a Three-Component Reaction For Trapping Kinase Substrate Complexes

Chemical Modification of Specific Active Site Amino Acid Residues of Enterobacter aerogenes Glycerol Dehydrogenase

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