Evidence for an Essential Lysine at the Active Site of <scp>l</scp>‐Histidinol : NAD<sup>+</sup> Oxidoreductase; a Bifunctional Dehydrogenase

Bürger, Erich; Görisch, Helmut

doi:10.1111/j.1432-1033.1981.tb05494.x

Cited by 19 publications

(12 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The result clearly demonstrates that the enzyme inactivation by PLP is solely due to the modification of Lys80. DISCUSSION PLP has been used to modify reactive lysine residues in various proteins including several NAD (P) + -dependent dehydrogenases (16)(17)(18)(19)(20)(21)(22), though the modifications are not necessarily active-site-directed (23). The inactivation by PLP of leucine dehydrogenase from B. sphaericus has also been reported, but the modified lysine residue (s) has not been identified (24).…”

Section: Construction Of Overexpression Plosmid and Purificationmentioning

confidence: 96%

Leucine Dehydrogenase from Bacillus stearothermophilus: Identification of Active-Site Lysine by Modification with Pyridoxal Phosphate1

Matsuyama

Soda

Fukui

et al. 1992

The Journal of Biochemistry

View full text Add to dashboard Cite

We have constructed an efficient expression plasmid for the leucine dehydrogenase gene previously cloned from Bacillus stearothermophilus. The recombinant enzyme was overproduced in Escherichia coli cells to a level of more than 30% of the total soluble protein upon induction with isopropyl beta-D-thiogalactopyranoside. The enzyme could be readily purified to homogeneity by heat treatment and a single step of ion-exchange chromatography. The purified enzyme was inactivated in a time-dependent manner upon incubation with pyridoxal 5'-phosphate (PLP) followed by reduction with sodium borohydride. The inactivation was completely prevented in the copresence of L-leucine and NAD+. Concomitantly with the inactivation, several molecules of PLP were incorporated into each subunit of the hexameric enzyme. Sequence analysis of the fluorescent peptides isolated from a proteolytic digest of the modified protein revealed that Lys80, Lys91, Lys206, and Lys265 were labeled. Among these residues, Lys80 was predominantly labeled and, in the presence of L-leucine and NAD+, was specifically protected from the labeling. Furthermore, a linear relationship of about 1:1 was observed between the extent of inactivation and the amount of PLP incorporated into Lys80. A slightly active mutant enzyme, in which Lys80 is replaced by Ala, was not inactivated at all by incubation with PLP, showing that the inactivation is correlated with the labeling of only Lys80. Lys80is conserved in the corresponding regions of all the amino acid dehydrogenase sequences reported to date. These results suggest that Lys80 is located at the active site and plays an important role in the catalytic function of leucine dehydrogenase.

show abstract

Section: Construction Of Overexpression Plosmid and Purificationmentioning

confidence: 96%

Leucine Dehydrogenase from Bacillus stearothermophilus: Identification of Active-Site Lysine by Modification with Pyridoxal Phosphate1

Matsuyama

Soda

Fukui

et al. 1992

The Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…To prevent decomposition of the unstable intermediate both oxidation steps are catalyzed by a single enzyme, histidinol dehydrogenase. The intermediate amino aldehyde is not released, which apparently is achieved by covalent bond formation between the aldehyde and an essential lysine at the active site [4].…”

Section: Discussionmentioning

confidence: 99%

“…The intermediate histidinal is firmly bound to the enzyme and cannot be trapped by carbonyl reagents [ 11. Histidinol dehydrogenase, however, will accept chemically synthesized histidinal and oxidize it to histidine in the presence of NAD' or in the reverse reaction of the first enzymatic step reduce it to histidinol with the concomitant oxidation of NADH [2, 31. Recently we demonstrated, that histidinol dehydrogenase possesses an essential lysine residue per subunit, which participates in the reversible oxidation -reduction reaction converting histidinal to histidinol [4]. An enzymatic reaction mechanism was proposed, in which oxidation of histidinol to histidinal is a concerted reaction involving the formation of a Schiff's base.…”

Section: Binding Of Histidinal To Histidinol Dehydrogenasementioning

confidence: 99%

“…NAD' and NADH were from Boehringer (Mannheim, FRG) and L-histidinol from Sigma (Munchen, FRG). L-Histidinal was synthesized according to Adams [5] as described previously [4]. Dedicated …”

Section: Chemicalsmentioning

confidence: 99%

“…Histidinol dehydrogenase was isolated from Salmonella typhimurium strain LT-2 his0 1242 [6], as described previously [4]. The purified enzyme routinely showed a specific activity of 12.5-13.5 units/mg when oxidizing histidinol at pH 9.4.…”

Section: Enzyme Preparationmentioning

confidence: 99%

See 2 more Smart Citations

Binding of histidinal to histidinol dehydrogenase

Görish

Hölke

1985

European Journal of Biochemistry

View full text Add to dashboard Cite

One molecule of the enzymatic intermediate histidinal is firmly bound per subunit of histidinol dehydrogenase (EC 1.1.1.23) and protected against decomposition. The dissociation rate constant of the histidinal–histidinol dehydrogenase complex is estimated as 2.5 × 10–5 S–1. Steady‐state kinetic measurements studying the oxidation of histidinol to histidine and the reduction of histidinal to histidinol allow to calculate the association rate constants for histidinal. For both reactions the association rate constant is found as 1.9 × 106 M–1 S–1. Thus the dissociation constant of the histidinal–histidinol dehydrogenase complex is estimated to be of the order of 1.4 × 10–11 M.

show abstract

Determination of pyridoxal‐5′‐phosphate (PLP)‐bonding sites in proteins: a peptide mass fingerprinting approach based on diagnostic tandem mass spectral features of PLP‐modified peptides

Simon¹,

Allison²

2009

Rapid Comm Mass Spectrometry

View full text Add to dashboard Cite

Peptides modified by pyridoxal-5'-phosphate (PLP), linked to a lysine residue via reductive amination, exhibit distinct spectral characteristics in the collision-induced dissociation (CID) tandem mass (MS/MS) spectra that are described here. The MS/MS spectra typically display two dominant peaks whose m/z values correspond to neutral losses of [H3PO4] (-98 Da) and the PLP moiety as [C8H10NO5P] (-231 Da) from the precursor peptide ion, respectively. Few other peaks are observed. Recognition of this distinct fragmentation behavior is imperative since determining sequences and sites of modifications relies on the formation of amide backbone cleavage products for subsequent interpretation via proteome database searching. Additionally, PLP-modified peptides exhibit suppressed precursor ionization efficiency which diminishes their detection in complex mixtures. Presented here is a protocol which describes an enrichment strategy for PLP-modified peptides combined with neutral loss screening and peptide mass fingerprinting to map the PLP-bonding site in a known PLP-dependent protein. This approach represents an efficient alternative to site-directed mutagenesis which has been the traditional method used for PLP-bonding site localization in proteins.

show abstract

Evidence for an Essential Lysine at the Active Site of l‐Histidinol : NAD⁺ Oxidoreductase; a Bifunctional Dehydrogenase

Cited by 19 publications

References 28 publications

Leucine Dehydrogenase from Bacillus stearothermophilus: Identification of Active-Site Lysine by Modification with Pyridoxal Phosphate1

Leucine Dehydrogenase from Bacillus stearothermophilus: Identification of Active-Site Lysine by Modification with Pyridoxal Phosphate1

Binding of histidinal to histidinol dehydrogenase

Determination of pyridoxal‐5′‐phosphate (PLP)‐bonding sites in proteins: a peptide mass fingerprinting approach based on diagnostic tandem mass spectral features of PLP‐modified peptides

Contact Info

Product

Resources

About

Evidence for an Essential Lysine at the Active Site of l‐Histidinol : NAD+ Oxidoreductase; a Bifunctional Dehydrogenase

Cited by 19 publications

References 28 publications

Leucine Dehydrogenase from Bacillus stearothermophilus: Identification of Active-Site Lysine by Modification with Pyridoxal Phosphate1

Leucine Dehydrogenase from Bacillus stearothermophilus: Identification of Active-Site Lysine by Modification with Pyridoxal Phosphate1

Binding of histidinal to histidinol dehydrogenase

Determination of pyridoxal‐5′‐phosphate (PLP)‐bonding sites in proteins: a peptide mass fingerprinting approach based on diagnostic tandem mass spectral features of PLP‐modified peptides

Contact Info

Product

Resources

About

Evidence for an Essential Lysine at the Active Site of l‐Histidinol : NAD⁺ Oxidoreductase; a Bifunctional Dehydrogenase