scholarly journals Inactivation of prolyl endopeptidase by a peptidylchloromethane. Kinetics of inactivation and identification of sites of modification

1991 ◽  
Vol 276 (3) ◽  
pp. 837-840 ◽  
Author(s):  
S R Stone ◽  
D Rennex ◽  
P Wikstrom ◽  
E Shaw ◽  
J Hofsteenge
Keyword(s):  
Active Site ◽  
Kinetic Mechanism ◽  
Cysteine Residue ◽  
Prolyl Endopeptidase ◽  
Histidine Residue ◽  
Progress Curve ◽  
Kinetics Of ◽  
Active Site Histidine

The kinetics of inactivation of prolyl endopeptidase by acetyl-Ala-Ala-Pro-CH2Cl were studied by progress-curve methods in the presence of substrate. The kinetic mechanism was found to involve the formation of an initial complex between the enzyme and the chloromethane followed by an inactivation step. The substrate was shown to compete for the formation of the initial complex, indicating that binding at the active site was a prerequisite for inactivation. After reaction of the enzyme with [3H]acetyl-Ala-Ala-Pro-CH2Cl, it was possible to isolate five labelled peptides. Four of these peptides contained a cysteine residue as the site of modification, whereas the fifth peptide contained no cysteine and a histidine residue was identified as the site of modification. This residue (His-680) probably represents the active-site histidine of prolyl endopeptidase.

scholarly journals Peptidyldiazomethanes. A novel mechanism of interaction with prolyl endopeptidase

1992 ◽  
Vol 283 (3) ◽  
pp. 871-876 ◽  
Author(s):  
S R Stone ◽  
D Rennex ◽  
P Wikstrom ◽  
E Shaw ◽  
J Hofsteenge
Keyword(s):  
Active Site ◽  
Kinetic Mechanism ◽  
Prolyl Endopeptidase ◽  
Binding Mechanism ◽  
Inhibitory Mechanism ◽  
Serine Residue ◽  
Mechanism Of Inhibition ◽  
Progress Curve ◽  
Mechanism Of Interaction ◽  
Kinetics Of

Peptidyldiazomethanes with proline in the P1 position were found to be competitive slow-binding inhibitors of prolyl endopeptidase. Progress-curve experiments monitoring the increase in the degree of inhibition with time indicated that the kinetic mechanism involved an initial complex that isomerized to form a tighter complex. Reversibility of the inhibited complex was demonstrated by monitoring the regain of enzyme activity after removal of free inhibitor and dilution into an assay containing competing substrate. The kinetics of the reversal of inhibition indicated a more complicated inhibitory mechanism involving more than one pathway for reversal of the tight complex. A slow-binding mechanism of inhibition has not been previously observed with peptidyldiazomethanes. Incorporation of [3H]Ac-Ala-Ala-Pro-diazomethane into prolyl endopeptidase was observed after denaturation of the inhibited complex. The peptide labelled with [3H]Ac-Ala-Ala-Pro-diazomethane was isolated and found to contain the active-site serine residue.

scholarly journals The location of the active-site histidine residue in the primary sequence of papain

1968 ◽  
Vol 108 (5) ◽  
pp. 861-866 ◽  
Author(s):  
S. S. Husain ◽  
G. Lowe
Keyword(s):  
Amino Acid ◽  
Sodium Borohydride ◽  
Active Site ◽  
Iodoacetic Acid ◽  
Cysteine Residue ◽  
Histidine Residue ◽  
Primary Sequence ◽  
Active Site Cysteine ◽  
Active Site Histidine ◽  
Active Site Cysteine Residue

Papain that had been irreversibly inhibited with 1,3-dibromo[2−14C]acetone was reduced with sodium borohydride and carboxymethylated with iodoacetic acid. After digestion with trypsin and α-chymotrypsin the radioactive peptides were purified chromatographically. Their amino acid composition indicated that cysteine-25 and histidine-106 were cross-linked. Since cysteine-25 is known to be the active-site cysteine residue, histidine-106 must be the active-site histidine residue.

Structural basis for the binding of succinate to succinyl-CoA synthetase

2016 ◽  
Vol 72 (8) ◽  
pp. 912-921 ◽  
Author(s):  
Ji Huang ◽  
Marie E. Fraser
Keyword(s):  
Citric Acid ◽  
Binding Site ◽  
Active Site ◽  
Citric Acid Cycle ◽  
Histidine Residue ◽  
Acid Cycle ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Active Site Histidine ◽  
Succinyl Coa Synthetase

Succinyl-CoA synthetase catalyzes the only step in the citric acid cycle that provides substrate-level phosphorylation. Although the binding sites for the substrates CoA, phosphate, and the nucleotides ADP and ATP or GDP and GTP have been identified, the binding site for succinate has not. To determine this binding site, pig GTP-specific succinyl-CoA synthetase was crystallized in the presence of succinate, magnesium ions and CoA, and the structure of the complex was determined by X-ray crystallography to 2.2 Å resolution. Succinate binds in the carboxy-terminal domain of the β-subunit. The succinate-binding site is near both the active-site histidine residue that is phosphorylated in the reaction and the free thiol of CoA. The carboxy-terminal domain rearranges when succinate binds, burying this active site. However, succinate is not in position for transfer of the phosphoryl group from phosphohistidine. Here, it is proposed that when the active-site histidine residue has been phosphorylated by GTP, the phosphohistidine displaces phosphate and triggers the movement of the carboxylate of succinate into position to be phosphorylated. The structure shows why succinyl-CoA synthetase is specific for succinate and does not react appreciably with citrate nor with the other C4-dicarboxylic acids of the citric acid cycle, fumarate and oxaloacetate, but shows some activity with L-malate.

scholarly journals Evidence for histidine in the active site of papain

1968 ◽  
Vol 108 (5) ◽  
pp. 855-859 ◽  
Author(s):  
S. S. Husain ◽  
G. Lowe
Keyword(s):  
Active Site ◽  
Tertiary Structure ◽  
Cysteine Residue ◽  
Optical Rotatory Dispersion ◽  
Performic Acid ◽  
Histidine Residue ◽  
Active Site Cysteine ◽  
Similar Amino Acid ◽  
Inhibited Enzyme ◽  
Active Site Cysteine Residue

Papain was irreversibly inhibited by 1,3-dibromoacetone, a reagent designed to react first with the active-site cysteine residue and subsequently with a second nucleophile. The molecular weight of the inhibited enzyme was indistinguishable from that of papain itself, and no evidence of dimeric or oligomeric species was found. The optical-rotatory-dispersion curves of chloroacetone-inhibited papain and 1,3-dibromoacetone-inhibited papain were essentially similar. Amino acid analysis of the 1,3-dibromo[2−14C]acetone-inhibited enzyme and the performic acid-oxidized material clearly showed that a cysteine and histidine residue had been alkylated through the thiol and N-1 of the imidazole group respectively. These groups must therefore be within 5å of each other in the tertiary structure of papain. Possible mechanistic implications are briefly discussed.

scholarly journals Tyrosine substitution of a conserved active-site histidine residue activates Plasmodium falciparum peroxiredoxin 6

2018 ◽  
Vol 28 (1) ◽  
pp. 100-110
Author(s):  
Kristina Feld ◽  
Fabian Geissel ◽  
Linda Liedgens ◽  
Robin Schumann ◽  
Sandra Specht ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Active Site ◽  
Histidine Residue ◽  
Peroxiredoxin 6 ◽  
Active Site Histidine
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