scholarly journals Identification of lysine at the active site of human 5-aminolaevulinate dehydratase

1986 ◽  
Vol 236 (2) ◽  
pp. 447-451 ◽  
Author(s):  
P N B Gibbs ◽  
P M Jordan
Keyword(s):  
Catalytic Activity ◽  
Amino Acid ◽  
Acid Hydrolysis ◽  
Active Site ◽  
Complete Loss ◽  
Enzyme Protein ◽  
Aminolaevulinic Acid ◽  
Cnbr Cleavage

Reduction of human 5-aminolaevulinate dehydratase with NaBH4 in the presence of 14C-labelled substrate led to complete loss of catalytic activity and to incorporation of label into the enzyme protein. By comparison with authentic lysyl-aminolaevulinic acid, prepared chemically, the modified active-site amino acid obtained by acid hydrolysis was shown to be lysine. Sequencing of a CNBr-cleavage peptide isolated from the inactivated 14C-labelled enzyme revealed that the lysine was present within the sequence M-V-K-P-G-M.

scholarly journals Prediction of the Evolution of Ceftazidime Resistance in Extended-Spectrum β-Lactamase CTX-M-9

2006 ◽  
Vol 50 (2) ◽  
pp. 731-738 ◽  
Author(s):  
J. Delmas ◽  
F. Robin ◽  
F. Carvalho ◽  
C. Mongaret ◽  
R. Bonnet
Keyword(s):  
Catalytic Activity ◽  
Amino Acid ◽  
Active Site ◽  
Random Mutagenesis ◽  
Evolutionary Process ◽  
Catalytic Efficiency ◽  
Kinetic Constants ◽  
The Other ◽  
Amino Acid Substitutions ◽  
Evolutionary Potential

ABSTRACT A random mutagenesis technique was used to predict the evolutionary potential of β-lactamase CTX-M-9 toward the acquisition of improved catalytic activity against ceftazidime. Thirty CTX-M mutants were obtained during three rounds of mutagenesis. These mutants conferred 1- to 128-fold-higher MICs of ceftazidime than the parental enzyme CTX-M-9. The CTX-M mutants contained one to six amino acid substitutions. Mutants harbored the substitutions Asp240Gly and Pro167Ser, which were previously observed in clinical CTX-M enzymes. Additional substitutions, notably Arg164His, Asp179Gly, and Arg276Ser, were observed near the active site. The kinetic constants of the three most active mutants revealed two distinct ways of improving catalytic efficiency against ceftazidime. One enzyme had a 17-fold-higher k cat value than CTX-M-9 against ceftazidime. The other two had 75- to 300-fold-lower Km values than CTX-M-9 against ceftazidime. The current emergence of CTX-M β-lactamases with improved activity against ceftazidime may therefore be the beginning of an evolutionary process which might subsequently generate a great diversity of CTX-M-type ceftazidimases.

scholarly journals Comparative Characterization of CTX-M-64 and CTX-M-14 Provides Insights into the Structure and Catalytic Activity of the CTX-M Class of Enzymes

2016 ◽  
Vol 60 (10) ◽  
pp. 6084-6090 ◽  
Author(s):  
Dandan He ◽  
Jiachi Chiou ◽  
Zhenling Zeng ◽  
Edward Wai-Chi Chan ◽  
Jian-Hua Liu ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Amino Acid ◽  
Active Site ◽  
Structural Integrity ◽  
Amino Acid Residues ◽  
Active Site Residues ◽  
Significant Difference ◽  
Function Relationship ◽  
Alignment Analysis ◽  
M 14

ABSTRACTClinical isolates producing hybrid CTX-M β-lactamases, presumably due to recombination between theblaCTX-M-15andblaCTX-M-14elements, have emerged in recent years. Among the hybrid enzymes, CTX-M-64 and CTX-M-14 display the most significant difference in catalytic activity. This study aims to investigate the mechanisms underlying such differential enzymatic activities in order to provide insight into the structure/function relationship of this class of enzymes. Sequence alignment analysis showed that the major differences between the amino acid composition of CTX-M-64 and CTX-M-14 lie at both the N and C termini of the enzymes. Single or multiple amino acid substitutions introduced into CTX-M-64 and CTX-M-14 were found to produce only minor effects on hydrolytic functions; such a finding is consistent with the notion that the discrepancy between the functional activities of the two enzymes is not the result of only a few amino acid changes but is attributable to interactions between a unique set of amino acid residues in each enzyme. This theory is supported by the results of the thermal stability assay, which confirmed that CTX-M-64 is significantly more stable than CTX-M-14. Our data confirmed that, in addition to the important residues located in the active site, residues distal to the active site also contribute to the catalytic activity of the enzyme through stabilizing its structural integrity.

scholarly journals Studies on the biotin-binding site of avidin. Lysine residues involved in the active site

1987 ◽  
Vol 242 (3) ◽  
pp. 923-926 ◽  
Author(s):  
G Gitlin ◽  
E A Bayer ◽  
M Wilchek
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Active Site ◽  
Amino Acid Analysis ◽  
Egg White ◽  
Reversed Phase ◽  
Complete Loss ◽  
Tryptic Peptides ◽  
Lysine Residues ◽  
Biotin Binding

Egg-white avidin was treated with 1-fluoro-2,4-dinitrobenzene. Modification of an average of one lysine residue per avidin subunit caused the complete loss of biotin binding. Tryptic peptides obtained from the 2,4-dinitrophenylated avidin were fractionated by reversed-phase h.p.l.c. Three peptides contained the 2,4-dinitrophenyl group. Amino acid analysis revealed that lysine residues 45, 94 and 111 are modified and probably comprise part of the biotin-binding site.

The amino acid sequence encompassing the active-site histidine residue of lipoamide dehydrogenase from Escherichia coli labelled with a bifunctional arsenoxide

1986 ◽  
Vol 64 (6) ◽  
pp. 509-514 ◽  
Author(s):  
Charles F. B. Holmes ◽  
Kenneth J. Stevenson
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
High Performance ◽  
Thiamine Pyrophosphate ◽  
Initial Reaction ◽  
Cnbr Cleavage ◽  
Lipoamide Dehydrogenase ◽  
Active Site Histidine ◽  
Inhibited Enzyme

Pyruvate dehydrogenase multienzyme complex (PD complex) in the presence of pyruvate, thiamine pyrophosphate, coenzyme A, and Mg2+ (or NADH) was irreversibly inhibited with the radiolabelled bifunctional arsenoxide p-[(bromoacetyl)-amino]phenyl arsenoxide (BrCH214CONHPhAsO). The initial reaction of the reagent was with a reduced lipoyl group of the lipoamide acetyltransferase component to form a dithioarsinite complex. Following the normal catalytic reactions, the anchored reagent was delivered into the active site of the lipoamide dehydrogenase (E3) component where an irreversible alkylation ensued via the bromoacetamidyl moiety. Treatment with 2,3-dithiopropanol (to break dithioarsinite bonds) caused the radio-labelled reagent to reside with E3. E3 was isolated from the inhibited PD complex and CNBr cleavage of the inhibited enzyme yielded a single radiolabelled peptide that was purified on a cyanopropyl silica column using high performance liquid chromatography. The radiolabelled amino acid was identified (after acid hydrolysis) as N3-[14C]carboxymethyl histidine in agreement with earlier studies. The radiolabel was located in residue 14 of the peptide for which the sequence was determined as[Formula: see text]This sequence agrees with the amino acid sequence determined from the gene sequence of E3. The histidine alkylated in the E3 component of the PD complex by BrCH214CONHPhAsO is residue-444 and further establishes its active site role.

scholarly journals Yeast phosphoglycerate mutate. Cyanogen bromide cleavage and amino acid sequence of an active-site peptide

1976 ◽  
Vol 153 (2) ◽  
pp. 145-149 ◽  
Author(s):  
L A Fothergill ◽  
G I Hodgson
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Cyanogen Bromide ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Phosphoglycerate Mutase ◽  
Large Fragment ◽  
X Ray ◽  
Cnbr Cleavage

The molecular weight and amino acid composition of phosphoglycerate mutase from yeast were determined. CNBr cleavage produced a large (190-residue) fragment and a small (60-residue) fragment. Tryptic and chymotryptic peptides derived from the large fragment were fractionated by ion-exchange chromatography. Peptides from two histidine-containing regions were isolated and the amino acid sequences were determined. Correlation of these data with X-ray-crystallographic evidence shows that the histidine residue in the sequence Arg-Leu Asn-Glu-Arg-His-Tyr-Gly-Asp-Leu-Glu-Gly-Lys is located at the active site.

Ni-S(uper)O(xide)D(ismutase) Inspired Ni(II)-Amino Acid Complexes Covalently Grafted onto Merrifield’s Resin - Synthesis, Structure and Catalytic Activity

2012 ◽  
Vol 730-732 ◽  
pp. 1012-1017
Author(s):  
Zita Csendes ◽  
Janos T. Kiss ◽  
Bence Kutus ◽  
Pal Sipos ◽  
Istvan Pálinkó
Keyword(s):  
Superoxide Dismutase ◽  
Catalytic Activity ◽  
Amino Acid ◽  
Active Site ◽  
Structural Features ◽  
Test Reaction ◽  
Sod Activity ◽  
X Ray ◽  
Amino Acid Complexes ◽  
Scanning Electron

In this work the syntheses of covalently grafted Ni(II)-complexes formed with various N- or C-protected amino acid ligands (L-histidine, L-tyrosine, L-cysteine and L-cystine) inspired by the active site of the Ni-SOD enzyme are presented. Merrifield’s resin was used as support to mimic the proteomic skeleton of the enzyme. Conditions of the syntheses were altered and the structural features of the substances obtained were studied by infrared spectroscopy. It was found that the preparation of covalently anchored Ni(II)−amino acid complexes was successful in all cases. In many cases the structures of the anchored complexes and the coordinating groups substantially varied upon changing the conditions of the syntheses. The obtained materials were studied by energy dispersive X-ray fluorescence coupled to scanning electron microscope (SEM−EDX). All the covalently anchored materials displayed superoxide dismutase (SOD) activity and some proved to be exceptionally efficient in the biochemical test reaction.

Amino acid sequence of cyanogen bromide fragment CB5 of human hemopexin

1989 ◽  
Vol 54 (3) ◽  
pp. 803-810 ◽  
Author(s):  
Ivan Kluh ◽  
Ladislav Morávek ◽  
Manfred Pavlík
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Acid Hydrolysis ◽  
Cyanogen Bromide ◽  
Polypeptide Chain ◽  
Amino Acid Residues ◽  
Mild Acid Hydrolysis ◽  
Methionine Residue ◽  
Cyanogen Bromide Fragment ◽  
Mild Acid

Cyanogen bromide fragment CB5 represents the region of the polypeptide chain of hemopexin between the fourth and fifth methionine residue (residues 232-352). It contains 120 amino acid residues in the following sequence: Arg-Cys-Ser-Pro-His-Leu-Val-Leu-Ser-Ala-Leu-Thr-Ser-Asp-Asn-His-Gly-Ala-Thr-Tyr-Ala-Phe-Ser-Gly-Thr-His-Tyr-Trp-Arg-Leu-Asp-Thr-Ser-Arg-Asp-Gly-Trp-His-Ser-Trp-Pro-Ile-Ala-His-Gln-Trp-Pro-Gln-Gly-Pro-Ser-Ala-Val-Asp-Ala-Ala-Phe-Ser-Trp-Glu-Glu-Lys-Leu-Tyr-Leu-Val-Gln-Gly-Thr-Gln-Val-Tyr-Val-Phe-Leu-Thr-Lys-Gly-Gly-Tyr-Thr-Leu-Val-Ser-Gly-Tyr-Pro-Lys-Arg-Leu-Glu-Lys-Glu-Val-Gly-Thr-Pro-His-Gly-Ile-Ile-Leu-Asp-Ser-Val-Asp-Ala-Ala-Phe-Ile-Cys-Pro-Gly-Ser-Ser-Arg-Leu-His-Ile-Met. The sequence was derived from the data on peptides prepared by cleavage of fragment CB5 by mild acid hydrolysis, by trypsin and chymotrypsin.

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