scholarly journals Inhibition of rabbit muscle aldolase by phosphorylated aromatic compounds

1997 ◽  
Vol 323 (1) ◽  
pp. 71-77 ◽  
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.

scholarly journals X-ray Crystallographic and Site-directed Mutagenesis Analysis of the Mechanism of Schiff-base Formation in Phosphonoacetaldehyde Hydrolase Catalysis

2003 ◽  
Vol 279 (10) ◽  
pp. 9353-9361 ◽  
Author(s):  
Marc C. Morais ◽  
Guofeng Zhang ◽  
Wenhai Zhang ◽  
David B. Olsen ◽  
Debra Dunaway-Mariano ◽  
...  
Keyword(s):  
Schiff Base ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
X Ray ◽  
Schiff Base Formation ◽  
Base Formation

scholarly journals Identification of the active-site lysine residues of two biosynthetic 3-dehydroquinases

1991 ◽  
Vol 275 (1) ◽  
pp. 1-6 ◽  
Author(s):  
S Chaudhuri ◽  
K Duncan ◽  
L D Graham ◽  
J R Coggins
Keyword(s):  
Escherichia Coli ◽  
Schiff Base ◽  
Nucleotide Sequence ◽  
Active Site ◽  
Active Sites ◽  
Amino Acid Sequences ◽  
E Coli ◽  
Lysine Residues ◽  
Schiff Base Formation ◽  
Base Formation

The lysine residues involved in Schiff-base formation at the active sites of both the 3-dehydroquinase component of the pentafunctional arom enzyme of Neurospora crassa and of the monofunctional 3-dehydroquinase of Escherichia coli were labelled by treatment with 3-dehydroquinate in the presence of NaB3H4. Radioactive peptides were isolated by h.p.l.c. following digestion with CNBr (and in one case after further digestion with trypsin). The sequence established for the N. crassa peptide was ALQHGDVVKLVVGAR, and that for the E. coli peptide was QSFDADIPKIA. An amended nucleotide sequence for the E. coli gene (aroD) that encode 3-dehydroquinase is also presented, along with a revised alignment of the deduced amino acid sequences for the biosynthetic enzymes.

scholarly journals Mechanistic Insights into the Allosteric Regulation of the Clr4 Protein Lysine Methyltransferase by Autoinhibition and Automethylation

2020 ◽  
Vol 21 (22) ◽  
pp. 8832
Author(s):  
Mina S. Khella ◽  
Alexander Bröhm ◽  
Sara Weirich ◽  
Albert Jeltsch
Keyword(s):  
Active Site ◽  
Histone H3 ◽  
Enzyme Activation ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Structural Studies ◽  
Peptide Substrates ◽  
Heterochromatin Formation ◽  
Lysine Residues ◽  
Lysine Methyltransferase

Clr4 is a histone H3 lysine 9 methyltransferase in Schizosaccharomyces pombe that is essential for heterochromatin formation. Previous biochemical and structural studies have shown that Clr4 is in an autoinhibited state in which an autoregulatory loop (ARL) blocks the active site. Automethylation of lysine residues in the ARL relieves autoinhibition. To investigate the mechanism of Clr4 regulation by autoinhibition and automethylation, we exchanged residues in the ARL by site-directed mutagenesis leading to stimulation or inhibition of automethylation and corresponding changes in Clr4 catalytic activity. Furthermore, we demonstrate that Clr4 prefers monomethylated (H3K9me1) over unmodified (H3K9me0) histone peptide substrates, similar to related human enzymes and, accordingly, H3K9me1 is more efficient in overcoming autoinhibition. Due to enzyme activation by automethylation, we observed a sigmoidal dependence of Clr4 activity on the AdoMet concentration, with stimulation at high AdoMet levels. In contrast, an automethylation-deficient mutant showed a hyperbolic Michaelis–Menten type relationship. These data suggest that automethylation of the ARL could act as a sensor for AdoMet levels in cells and regulate the generation and maintenance of heterochromatin accordingly. This process could connect epigenome modifications with the metabolic state of cells. As other human protein lysine methyltransferases (for example, PRC2) also use automethylation/autoinhibition mechanisms, our results may provide a model to describe their regulation as well.

scholarly journals Function of the 90-loop (Thr90–Glu100) region of staphylokinase in plasminogen activation probed through site-directed mutagenesis and loop deletion

2002 ◽  
Vol 365 (2) ◽  
pp. 379-389 ◽  
Author(s):  
Govindan RAJAMOHAN ◽  
Monika DAHIYA ◽  
Shekhar C. MANDE ◽  
Kanak L. DIKSHIT
Keyword(s):  
Active Site ◽  
Complex Function ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Kringle Domains ◽  
Lysine Residues ◽  
Modelling Studies ◽  
Activator Complex

Staphylokinsae (SAK) forms a bimolecular complex with human plasmin(ogen) and changes its substrate specificity by exposing new exosites that enhances accession of substrate plasminogen (PG) to the plasmin (Pm) active site. Protein modelling studies indicated the crucial role of a loop in SAK (SAK 90-loop; Thr90—Glu100) for the docking of the substrate PG to the SAK—Pm complex. Function of SAK 90-loop was studied by site-directed mutagenesis and loop deletion. Deletion of nine amino acid residues (Tyr92—Glu100) from the SAK 90-loop, resulted in ≈60% reduction in the PG activation, but it retained the ability to generate an active site within the complex of loop mutant of SAK (SAKΔ90) and Pm. The preformed activator complex of SAKΔ90 with Pm, however, displayed a 50–60% reduction in substrate PG activation that remained unaffected in the presence of kringle domains (K1+K2+K3+K4) of PG, whereas PG activation by SAK—Pm complex displayed ∼50% reduction in the presence of kringles, suggesting the involvement of the kringle domains in modulating the PG activation by native SAK but not by SAKΔ90. Lysine residues (Lys94, Lys96, Lys97 and Lys98) of the SAK 90-loop were individually mutated into alanine and, among these four SAK loop mutants, SAKK97A and SAKK98A exhibited specific activities about one-third and one-quarter respectively of the native SAK. The kinetic parameters of PG activation of their 1:1 complex with Pm indicated that the Km values of PG towards the activator complex of these two SAK mutants were 4–6-fold higher, suggesting the decreased accessibility of the substrate PG to the activator complex formed by these SAK mutants. These results demonstrated the involvement of the Lys97 and Lys98 residues of the SAK 90-loop in assisting the interaction with substrate PG. These interactions of SAK—Pm activator complex via the SAK 90-loop may provide additional anchorage site(s) to the substrate PG that, in turn, may promote the overall process of SAK-mediated PG activation.

Schiff Base Formation upon Complexation of Tris(acetylacetonato)iron with N[(CH2CH2NH2)(CH2CH2OH)(CH2CH2CH2OH)]

2004 ◽  
Vol 630 (13-14) ◽  
pp. 2558-2561 ◽  
Author(s):  
F. Ekkehardt Hahn ◽  
Christoph Jocher ◽  
Thomas L�gger ◽  
Tania Pape
Keyword(s):  
Schiff Base ◽  
Schiff Base Formation ◽  
Base Formation
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