Modification of pig M4 lactate dehydrogenase by pyridoxal 5'-phosphate. Demonstration of an essential lysine residue

1. Pig M4 lactate dehydrogenase treated in the dark with pyridoxal 5'-phosphate at pH8.5 and 25 degrees C loses activity gradually. The maximum inactivation was 66%, and this did not increase with concentrations of pyridoxal 5'-phosphate above 1 mM. 2. Inactivation may be reversed by dialysis or made permanent by reducing the enzyme with NaBH4. 3. Spectral evidence indicates modification of lysine residues, and 6-N-pyridoxyl-lysine is present in the hydrolsate of inactivated, reduced enzyme. 4. A second cycle of treatment with pyridoxal 5'-phosphate and NaBH4 further decreases activity. After three cycles only 9% of the original activity remains. 5. Apparent Km values for lactate and NAD+ are unaltered in the partially inactivated enzyme. 6. These results suggest that the covalently modified enzyme is inactive; failure to achieve complete inactivation in a single treatment is due to the reversibility of Schiff-base formation and to the consequent presence of active non-covalently bonded enzyme-modifier complex in the equilibrium mixture. 7. Although several lysine residues per subunit are modified, only one appears to be essential for activity: pyruvate and NAD+ together (both 5mM) completely protect against inactivation, and there is a one-to-one relationship between enzyme protection and decreased lysine modification. 8. NAD+ or NADH alone gives only partial protection. Substrates give virtually none. 9. Pig H4 lactate dehydrogenase is also inactivated by pyridoxal 5'-phosphate. 10. The possible role of the essential lysine residue is discussed.

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The Action of Thrombin on Modified Fibrinogen

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657364 ◽

1983 ◽

Vol 49 (03) ◽

pp. 208-213

Author(s):

A J Osbahr

Keyword(s):

Physical Properties ◽

Negative Charge ◽

Lysine Residue ◽

Fibrinopeptide A ◽

Amino Groups ◽

Lysine Residues ◽

Citraconic Anhydride

SummaryThe modification of canine fibrinogen with citraconic anhydride modified the ε-amino groups of the fibrinogen and at the same time generated additional negative charges into the protein. The addition of thrombin to the modified fibrinogen did not induce polymerization; however, the fibrinopeptide was released at a faster rate than from the unmodified fibrinogen. The physical properties of the citraconylated fibrinogen were markedly altered by the modification of 50-60 lysine residues in one hour. A modified fibrinopeptide-A was released by thrombin from the modified fibrinogen and was electrophoretically more anionic than the unmodified fibrinopeptide-A. Edman analysis confirmed the modification of the lysine residue present in the peptide. The rate of removal of citraconylated fibrinopeptide-A from modified fibrinogen by thrombin was 30 to 40 percent greater than the cleavage of unmodified fibrinopeptide-A from unmodified fibrinogen. However, the modification of 60 or more lysine residues in the fibrinogen produced a decrease in the rate of cleavage of citraconylated fibrinopeptide-A. The results suggest that additional negative charge in the vicinity of the attachment of fibrinopeptide-A to canine fibrinogen aids in the removal of the peptide by thrombin.

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Small Mass but Strong Information: Diagnostic Ions Provide Crucial Clues to Correctly Identify Histone Lysine Modifications

Proteomes ◽

10.3390/proteomes9020018 ◽

2021 ◽

Vol 9 (2) ◽

pp. 18

Author(s):

Alaa Hseiky ◽

Marion Crespo ◽

Sylvie Kieffer-Jaquinod ◽

François Fenaille ◽

Delphine Pflieger

Keyword(s):

Amino Acid ◽

Small Mass ◽

Mass Region ◽

Amino Acid Sequences ◽

Lysine Modification ◽

Lysine Residues ◽

Two Factors ◽

Low Mass ◽

Diagnostic Ions

(1) Background: The proteomic analysis of histones constitutes a delicate task due to the combination of two factors: slight variations in the amino acid sequences of variants and the multiplicity of post-translational modifications (PTMs), particularly those occurring on lysine residues. (2) Methods: To dissect the relationship between both aspects, we carefully evaluated PTM identification on lysine 27 from histone H3 (H3K27) and the artefactual chemical modifications that may lead to erroneous PTM determination. H3K27 is a particularly interesting example because it can bear a range of PTMs and it sits nearby residues 29 and 31 that vary between H3 sequence variants. We discuss how the retention times, neutral losses and immonium/diagnostic ions observed in the MS/MS spectra of peptides bearing modified lysines detectable in the low-mass region might help validate the identification of modified sequences. (3) Results: Diagnostic ions carry key information, thereby avoiding potential mis-identifications due to either isobaric PTM combinations or isobaric amino acid-PTM combinations. This also includes cases where chemical formylation or acetylation of peptide N-termini artefactually occurs during sample processing or simply in the timeframe of LC-MS/MS analysis. Finally, in the very subtle case of positional isomers possibly corresponding to a given mass of lysine modification, the immonium and diagnostic ions may allow the identification of the in vivo structure.

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Horse liver alcohol dehydrogenase. A study of the essential lysine residue

Biochemical Journal ◽

10.1042/bj1490627 ◽

1975 ◽

Vol 149 (3) ◽

pp. 627-635 ◽

Cited By ~ 17

Author(s):

S S Chen ◽

P C Engel

Keyword(s):

Alcohol Dehydrogenase ◽

Rate Constant ◽

Lysine Residue ◽

Horse Liver Alcohol Dehydrogenase ◽

First Order ◽

Liver Alcohol Dehydrogenase ◽

Horse Liver ◽

Lysine Residues ◽

Modified Enzyme ◽

Covalent Complex

1. The inactivation of horse liver alcohol dehydrogenase by pyridoxal 5'-phosphate in phosphate buffer, pH8, at 10°C was investigated. Activity declines to a minimum value determined by the pyridoxal 5'-phosphate concentration. The maximum inactivation in a single treatment is 75%. This limit appears to be set by the ratio of the first-order rate constants for interconversion of inactive covalently modified enzyme and a readily dissociable non-covalent enzyme-modifier complex. 2. Reactivation was virtually complete on 150-fold dilution: first-order analysis yielded an estimate of the rate constant (0.164min-1), which was then used in the kinetic analysis of the forward inactivation reaction. This provided estimates for the rate constant for conversion of non-covalent complex into inactive enzyme (0.465 min-1) and the dissociation constant of the non-covalent complex (2.8 mM). From the two first-order constants, the minimum attainable activity in a single cycle of treatment may be calculated as 24.5%, very close to the observed value. 3. Successive cycles of modification followed by reduction with NaBH4 each decreased activity by the same fraction, so that three cycles with 3.6 mM-pyridoxal 5'-phosphate decreased specific activity to about 1% of the original value. The absorption spectrum of the enzyme thus treated indicated incorporation of 2-3 mol of pyridoxal 5'-phosphate per mol of subunit, covalently bonded to lysine residues. 4. NAD+ and NADH protected the enzyme completely against inactivation by pyridoxal 5'-phosphate, but ethanol and acetaldehyde were without effect. 5. Pyridoxal 5'-phosphate used as an inhibitor in steady-state experiments, rather than as an inactivator, was non-competitive with respect to both NADH and acetaldehyde. 6. The partially modified enzyme (74% inactive) showed unaltered apparent Km values for NAD+ and ethanol, indicating that modified enzyme is completely inactive, and that the residual activity is due to enzyme that has not been covalently modified. 7. Activation by methylation with formaldehyde was confirmed, but this treatment does not prevent subsequent inactivation with pyridoxal 5'-phosphate. Presumably different lysine residues are involved. 8. It is likely that the essential lysine residue modified by pyridoxal 5'-phosphate is involved either in binding the coenzymes or in the catalytic step. 9. Less detailed studies of yeast alcohol dehydrogenase suggest that this enzyme also possesses an essential lysine residue.

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Chemical Footprinting Reveals Conformational Changes Following Activation of Factor XI

Thrombosis and Haemostasis ◽

10.1160/th17-09-0676 ◽

2018 ◽

Vol 118 (02) ◽

pp. 340-350 ◽

Cited By ~ 3

Author(s):

Ingrid Stroo ◽

J. Marquart ◽

Kamran Bakhtiari ◽

Tom Plug ◽

Alexander Meijer ◽

...

Keyword(s):

Conformational Change ◽

Conformational Changes ◽

Catalytic Domain ◽

Active Form ◽

Coagulation Factor ◽

Lysine Residue ◽

Factor Ix ◽

Factor Xi ◽

Lysine Residues ◽

Factor Xia

AbstractCoagulation factor XI is activated by thrombin or factor XIIa resulting in a conformational change that converts the catalytic domain into its active form and exposing exosites for factor IX on the apple domains. Although crystal structures of the zymogen factor XI and the catalytic domain of the protease are available, the structure of the apple domains and hence the interactions with the catalytic domain in factor XIa are unknown. We now used chemical footprinting to identify lysine residue containing regions that undergo a conformational change following activation of factor XI. To this end, we employed tandem mass tag in conjunction with mass spectrometry. Fifty-two unique peptides were identified, covering 37 of the 41 lysine residues present in factor XI. Two identified lysine residues that showed altered flexibility upon activation were mutated to study their contribution in factor XI stability or enzymatic activity. Lys357, part of the connecting loop between A4 and the catalytic domain, was more reactive in factor XIa but mutation of this lysine residue did not impact on factor XIa activity. Lys516 and its possible interactor Glu380 are located in the catalytic domain and are covered by the activation loop of factor XIa. Mutating Glu380 enhanced Arg369 cleavage and thrombin generation in plasma. In conclusion, we have identified novel regions that undergo a conformational change following activation. This information improves knowledge about factor XI and will contribute to development of novel inhibitors or activators for this coagulation protein.

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Comparison of the activity and conformation changes of lactate dehydrogenase H4 during denaturation by guanidinium chloride

Biochemical Journal ◽

10.1042/bj2770207 ◽

1991 ◽

Vol 277 (1) ◽

pp. 207-211 ◽

Cited By ~ 42

Author(s):

Y Z Ma ◽

C L Tsou

Keyword(s):

Lactate Dehydrogenase ◽

Active Site ◽

Cross Linking ◽

Native Conformation ◽

Guanidinium Chloride ◽

Original Activity ◽

Limited Region ◽

Low Concentrations ◽

Two Stages ◽

Inhibited Enzyme

The inactivation and unfolding of lactate dehydrogenase (LDH) during denaturation by guanidinium chloride (GuHCl) under diverse conditions have been compared. Unfolding of the native conformation, as monitored by fluorescence and c.d. measurements, occurs in two stages with increasing GuHCl concentrations, and the inactivation approximately coincides with, but slightly precedes, the first stage of unfolding. The enzyme is inhibited to about 60-70% of its original activity by cross-linking with glutaraldehyde or in the presence of 1 M-(NH4)2SO4, with its conformation stabilized as shown by the requirement for higher GuHCl concentrations to bring about both inactivation and unfolding. Low concentrations of GuHCl (0.2-0.4 M) activate the cross-linked and the (NH4)2SO4-inhibited enzyme back to the level of the native enzyme. For the enzyme stabilized by (NH4)2SO4 or by cross-linking with glutaraldehyde, inactivation occurs at a markedly lower GuHCl concentration than that required to bring about its first stage of unfolding. It is concluded that the active site of LDH is situated in a limited region relatively fragile in conformation as compared with the molecule as a whole. The GuHCl activation of LDH stabilized in (NH4)2SO4 or by cross-linking with glutaraldehyde suggests that this fragility and consequently flexibility of the active site is required for its catalytic activity.

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Role of positive charge of lysine residue on cytochrome c3 for electrostatic interaction with hydrogenase

Journal of Porphyrins and Phthalocyanines ◽

10.1142/s1088424607000096 ◽

2007 ◽

Vol 11 (01) ◽

pp. 66-73

Author(s):

Shin Iida ◽

Noriyuki Asakura ◽

Kenji Tabata ◽

Ichiro Okura ◽

Toshiaki Kamachi

Keyword(s):

Electron Transfer ◽

Cytochrome C ◽

Hydrogen Evolution ◽

Electrostatic Interaction ◽

Positive Charge ◽

Lysine Residue ◽

Site Directed Mutagenesis ◽

Desulfovibrio Vulgaris ◽

Lysine Residues

Cytochrome c3 from Desulfovibrio vulgaris (Miyazaki) is an electron transfer protein containing four hemes per molecule. Its physiological electron transfer partner is the hydrogenase which catalyzes reversible oxidation of hydrogen. The complex formation between cytochrome c3 and hydrogenase is caused by electrostatic interaction, because cytochrome c3 is a basic protein and hydrogenase is an acidic protein. As cytochrome c3 has 20 lysine residues among 108 amino acids, the positive charges of some lysine residues may play an important role in the interaction with hydrogenase. To clarify the role of positive charge of lysine residue, the positive charge was changed to neutral or negative charge using chemical modification and site-directed mutagenesis. When the positive charges around heme IV were changed, the hydrogen evolution rate with hydrogenase decreased. The affinity between hydrogenase and mutated cytochrome c3 (K57Q, K57E, K72Q, K94Q, K94E) were not affected. On the other hand, the affinity of K72E cytochrome c3 for hydrogenase was very low. These results suggest that the positive charge around heme IV plays an important role in the electrostatic interaction with hydrogenase in hydrogen evolution.

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Modification of hydroxymethylbilane synthase (porphobilinogen deaminase) by pyridoxal 5′-phosphate. Demonstration of an essential lysine residue

Biochemical Journal ◽

10.1042/bj2220093 ◽

1984 ◽

Vol 222 (1) ◽

pp. 93-102 ◽

Cited By ~ 20

Author(s):

G J Hart ◽

F J Leeper ◽

A R Battersby

Keyword(s):

Euglena Gracilis ◽

Enzymic Activity ◽

Lysine Residue ◽

Porphobilinogen Deaminase ◽

Amino Group ◽

Spectroscopic Evidence ◽

Lysine Residues ◽

Modified Enzyme ◽

Rhodopseudomonas Spheroides ◽

Covalently Bound

When hydroxymethylbilane synthase (porphobilinogen deaminase) from Euglena gracilis is incubated with pyridoxal 5′-phosphate at pH 7.0 and 0 degree C, it rapidly loses part of its activity. The proportion of activity that remains decreases as the concentration of the modifier increases up to approx. 2mM, above which no further significant inactivation occurs. Dialysis of the partly inactivated enzyme restores its activity, whereas reduction with NaBH4 makes the inactivation permanent. The maximum inactivation achievable from one cycle of the treatment with pyridoxal 5′-phosphate, then with borohydride, is 53 +/- 5%; taking this modified enzyme through second and third cycles causes further loss of activity. The enzyme from Rhodopseudomonas spheroides behaves similarly, but there are quantitative differences. Spectroscopic evidence indicates that the inactivation procedure modifies lysine residues, and labelling studies show that epsilon-N-pyridoxyl-L-lysine is a product when permanently inactivated enzyme is completely hydrolysed. Several lysine residues per molecule of the E. gracilis enzyme are modified by the treatment with pyridoxal 5′-phosphate and borohydride, but only one appears to be essential for enzymic activity, since porphobilinogen protects the enzyme against inactivation and then one fewer lysine residue per molecule of enzyme is affected. It is suggested that, during the biosynthesis of hydroxymethylbilane, the first porphobilinogen unit is covalently bound to the enzyme through the epsilon-amino group of the essential lysine.

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Inhibition of rabbit muscle aldolase by phosphorylated aromatic compounds

Biochemical Journal ◽

10.1042/bj3230071 ◽

1997 ◽

Vol 323 (1) ◽

pp. 71-77 ◽

Cited By ~ 23

Author(s):

Casimir BLONSKI ◽

Danielle DE MOISSAC ◽

Jacques PÉRIÉ ◽

Jurgen SYGUSCH

Keyword(s):

Schiff Base ◽

Active Site ◽

Enzyme Inactivation ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Rabbit Muscle ◽

Lysine Residues ◽

Schiff Base Formation ◽

Fructose 1,6 Bisphosphate ◽

Base Formation

The interactions of the phosphorylated derivatives of hydroquinone (HQN-P2), resorcinol (RSN-P2), 4-hydroxybenzaldehyde (HBA-P) and 2,4-dihydroxybenzaldehyde (DHBA-P; phosphate group at position 4) with fructose bisphosphate aldolase were analysed by enzyme kinetics, UV/visible difference spectroscopy and site-directed mutagenesis. Enzyme activity was competitively inhibited in the presence of HQN-P2, RSN-P2 and HBA-P, whereas DHBA-P exhibited slow-binding inhibition. Inhibition by DHBA-P involved active-site Schiff-base formation and required a phenol group ortho to the aldehyde moiety. Rates of enzyme inactivation and of Schiff-base formation by DHBA-P were identical, and corresponded to 3.2-3.5 DHBA-P molecules covalently bound per aldolase tetramer at maximal inactivation. Site-directed mutagenesis of the active-site lysine residues at positions 107, 146 and 229 was found to be consistent with Schiff-base formation between DHBA-P and Lys-146, and this was promoted by Lys-229. Mutation of Glu-187, located vicinally between Lys-146 and Lys-229 in the active site, perturbed the rate of Schiff-base formation, suggesting a functional role for Glu-187 in Schiff-base formation and stabilization. The decreased cleavage activity of the active-site mutants towards fructose 1,6-bisphosphate is consistent with a proton-transfer mechanism involving Lys-229, Glu-187 and Lys-146.

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Hydroxylammonium derivatives for selective active-site lysine modification in the anti-virulence bacterial target DHQ1 enzyme

Organic Chemistry Frontiers ◽

10.1039/c9qo00453j ◽

2019 ◽

Vol 6 (17) ◽

pp. 3127-3135 ◽

Cited By ~ 1

Author(s):

María Maneiro ◽

Emilio Lence ◽

Marta Sanz-Gaitero ◽

José M. Otero ◽

Mark J. van Raaij ◽

...

Keyword(s):

Drug Discovery ◽

Active Site ◽

Covalent Modification ◽

Lysine Residue ◽

Lysine Modification ◽

Promising Target

The first example of a hydroxylammonium derivative that causes a specific covalent modification of the active-site lysine residue of an aldolase enzyme, which is a promising target for anti-bacterial drug discovery, is reported.

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Affinity labelling of the NADP+-binding site of glucose 6-phosphate dehydrogenase from Candida utilis

Biochemical Journal ◽

10.1042/bj1830297 ◽

1979 ◽

Vol 183 (2) ◽

pp. 297-302 ◽

Cited By ~ 16

Author(s):

T Bellini ◽

M Signorini ◽

F Dallocchio ◽

M Rippa

Keyword(s):

Binding Site ◽

Candida Utilis ◽

Aldehyde Group ◽

Lysine Residue ◽

Reversible Inactivation ◽

Coenzyme Binding ◽

Complete Inactivation ◽

Affinity Labelling ◽

Glucose 6 Phosphate Dehydrogenase ◽

Coenzyme Binding Site

1. Periodate-oxidized NADP+ inhibits the catalytic activity of glucose 6-phosphate dehydrogenase from Candida utilis, competing with NADP+. 2. Incubation of the enzyme with the coenzyme analogue causes partial reversible inactivation of the enzyme as a result of affinity labelling of the coenzyme-binding site. 3. Some kinetic values of the reaction were calculated. 4. The inactivation can be made irreversible by treatment with NaBH4, which reduces a Schiff base formed between an aldehyde group on the coenzyme analogue and a lysine residue on the enzyme. 5. Complete inactivation can be correlated with the binding of only one inhibitor to each enzyme subunit. 6. The lysine residue involved in the binding of the inhibitor is present at the coenzyme-binding site.

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