scholarly journals A reinvestigation of residues 64–68 and 175 in papain. Evidence that residues 64 and 175 are asparagine

1970 ◽  
Vol 116 (4) ◽  
pp. 689-692 ◽  
Author(s):  
S. S. Husain ◽  
G. Lowe
Keyword(s):  
Active Site ◽  
Tertiary Structure ◽  
Thiol Group ◽  
Imidazole Ring ◽  
Histidine Residue ◽  
Active Site Cysteine ◽  
Density Map ◽  
A Site ◽  
Electron Density Map ◽  
Active Site Histidine

The tryptophan-containing peptides were isolated from the chymotryptic digest of S-carboxymethylated papain. Residue 175, which is strongly hydrogen-bonded to the active-site histidine residue in the tertiary structure of papain, is asparagine, confirming the work of Kimmel, Rogers & Smith (1965). Its function is probably to maintain the orientation and tautomeric state of the imidazole ring of histidine-159. The amino acid sequence predicted from the electron-density map of papain for residues 64–68 was confirmed, but residue 64 is asparagine, not aspartic acid. This residue, which is about 10 Å from the thiol group of the active-site cysteine-25, cannot therefore be a site of electrostatic attraction for substrates of basic amino acids.

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.

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 Determination of the ionization state of the active-site histidine in a subtilisin-(chloromethane inhibitor) derivative by 13C-NMR

1996 ◽  
Vol 317 (1) ◽  
pp. 35-40 ◽  
Author(s):  
Timothy P. O'CONNELL ◽  
J. Paul G. MALTHOUSE
Keyword(s):  
13C Nmr ◽  
Active Site ◽  
Catalytic Mechanism ◽  
13C Nmr Spectroscopy ◽  
Imidazole Ring ◽  
Histidine Residue ◽  
Ionization State ◽  
Methylene Carbon ◽  
Active Site Histidine

Subtilisin BPN´ has been alkylated using benzyloxycarbonylglycylglycyl[1-13C]phenylalanylchloromethane. Using difference 13C-NMR spectroscopy a single signal due to the 13C-enriched α-methylene carbon of the subtilisin–(chloromethane inhibitor) derivative was detected. No evidence for the denaturation/autolysis of this derivative was obtained from pH 3.5 to 11.5. However, incubating at pH 12.75 or heating in the presence of SDS at pH 6.9 did denature this derivative. The negative titration shift of the α-methylene carbon of the denatured derivatives confirmed that the inhibitor had alkylated N-3 of the imidazole ring of the active-site histidine. The positive titration shift of 3.96 p.p.m. and the pKa of 7.04 obtained from studying the native subtilisin–(chloromethane inhibitor) derivative are assigned to oxyanion formation. We conclude that the pKa of the alkylated histidine residue in the native subtilisin–(chloromethane inhibitor) derivative must be > 12 and that subtilisin preferentially stabilizes the zwitterionic tetrahedral adduct consisting of the oxyanion and the imidazolium ion of the active-site histidine residue. We show that even before the oxyanion is formed the pKa of the active-site histidine must be much greater than that of the oxyanion in the zwitterionic tetrahedral adduct. We discuss the significance of our results for the catalytic mechanism of the serine proteinases.

scholarly journals Chemical modification of serine at the active site of penicillin acylase from Kluyvera citrophila

1991 ◽  
Vol 280 (3) ◽  
pp. 659-662 ◽  
Author(s):  
J Martín ◽  
A Slade ◽  
A Aitken ◽  
R Arche ◽  
R Virden
Keyword(s):  
Active Site ◽  
Tertiary Structure ◽  
Thiol Group ◽  
Iodoacetic Acid ◽  
Penicillin Acylase ◽  
Protein Product ◽  
Side Chain ◽  
Serine Residue ◽  
Conserved Sequence ◽  
Ester Substrate

The site of reaction of penicillin acylase from Kluyvera citrophila with the potent inhibitor phenylmethanesulphonyl fluoride was investigated by incubating the inactivated enzyme with thioacetic acid to convert the side chain of the putative active-site serine residue to that of cysteine. The protein product contained one thiol group, which was reactive towards 2,2′-dipyridyl disulphide and iodoacetic acid. Carboxymethylcysteine was identified as the N-terminal residue of the beta-subunit of the carboxy[3H]methylthiol-protein. No significant changes in tertiary structure were detected in the modified penicillin acylase using near-u.v. c.d. spectroscopy. However, the catalytic activity (kcat) with either an anilide or an ester substrate was decreased in the thiol-protein by a factor of more than 10(4). A comparison of sequences of apparently related acylases shows no other extensive regions of conserved sequence containing an invariant serine residue. The side chain of this residue is proposed as a candidate nucleophile in the formation of an acyl-enzyme during catalysis.

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 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.

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