Evidence for Nonproductive Binding Subsites within the Active Site of Papain

1974 ◽  
Vol 52 (10) ◽  
pp. 877-883 ◽  
Author(s):  
Bernard R. Glick ◽  
Lewis J. Brubacher
Keyword(s):  
Ethyl Ester ◽  
Rate Constant ◽  
Active Site ◽  
Alkylating Agents ◽  
Sulfhydryl Group ◽  
Competitive Inhibitor ◽  
Alkylating Reagents ◽  
Nonproductive Binding ◽  
Binding Subsites

The reactions of several alkylating reagents with the sulfhydryl group in papain have been studied in the presence of varying concentrations of the competitive inhibitor α-N-benzoyl-D-arginine ethyl ester. The ratio of the alkylation rate constant of the papain – α-N-benzoyl-D-arginine ethyl ester complex to the rate constant with free papain is 4.3, 1.2, and 0.0 for the alkylating agents 1-chloro-3-tosylamido-4-phenyl-2-butanone, N-ethylmaleimide, and 1-chloro-3-tosylamido-7-amino-2-heptanone, respectively. These results are rationalized, along with data for the effect of α-N-benzoyl-L-arginine ethyl ester, in terms of nonproductive binding.

scholarly journals Role of Arginines in Coenzyme A Binding and Catalysis by the Phosphotransacetylase from Methanosarcina thermophila

2001 ◽  
Vol 183 (14) ◽  
pp. 4244-4250 ◽  
Author(s):  
Prabha P. Iyer ◽  
James G. Ferry
Keyword(s):  
Active Site ◽  
Coenzyme A ◽  
Sulfhydryl Group ◽  
Salt Bridge ◽  
Competitive Inhibitor ◽  
Enzyme Inactivation ◽  
Methanosarcina Thermophila ◽  
Arginine Residues ◽  
Substrate Protection

ABSTRACT Phosphotransacetylase (EC 2.3.1.8 ) catalyzes the reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA): CH3COOPO3 2− + CoASH ⇆ CH3COSCoA + HPO4 2−. The role of arginine residues was investigated for the phosphotransacetylase from Methanosarcina thermophila. Kinetic analysis of a suite of variants indicated that Arg 87 and Arg 133 interact with the substrate CoA. Arg 87 variants were reduced in the ability to discriminate between CoA and the CoA analog 3′-dephospho-CoA, indicating that Arg 87 forms a salt bridge with the 3′-phosphate of CoA. Arg 133 is postulated to interact with the 5′-phosphate of CoA. Large decreases in k cat andk cat/Km for all of the Arg 87 and Arg 133 variants indicated that these residues are also important, although not essential, for catalysis. Large decreases ink cat andk cat/Km were also observed for the variants in which lysine replaced Arg 87 and Arg 133, suggesting that the bidentate interaction of these residues with CoA or their greater bulk is important for optimal activity. Desulfo-CoA is a strong competitive inhibitor of the enzyme, suggesting that the sulfhydryl group of CoA is important for the optimization of CoA-binding energy but not for tight substrate binding. Chemical modification of the wild-type enzyme by 2,3-butanedione and substrate protection by CoA indicated that at least one reactive arginine is in the active site and is important for activity. The inhibition pattern of the R87Q variant indicated that Arg 87 is modified, which contributes to the inactivation; however, at least one additional active-site arginine is modified leading to enzyme inactivation, albeit at a lower rate.

scholarly journals Evidence for the importance of arginine residues in pig kidney alkaline phosphatase

1979 ◽  
Vol 181 (1) ◽  
pp. 137-142 ◽  
Author(s):  
M N Woodroofe ◽  
P J Butterworth
Keyword(s):  
Alkaline Phosphatase ◽  
Active Site ◽  
Competitive Inhibitor ◽  
Arginine Residue ◽  
Pig Kidney ◽  
Close Proximity ◽  
Arginine Residues ◽  
Km Value ◽  
Uncompetitive Inhibitor ◽  
Kidney Alkaline Phosphatase

The arginine-specific reagents 2,3-butanedione and phenylglyoxal inactivate pig kidney alkaline phosphatase. As inactivation proceeds there is a progressive fall in Vmax. of the enzyme, but no demonstrable change in the Km value for substrate. Pi, a competitive inhibitor, and AMP, a substrate of the enzyme, protect alkaline phosphatase against the arginine-specific reagents. These effects are explicable by the assumption that the enzyme contains an essential arginine residue at the active site. Protection is also afforded by the uncompetitive inhibitor NADH through a partially competive action against the reagents. Enzyme that has been exposed to the reagents has a decreased sensitivity to NADH inhibition. It is suggested that an arginine residue is important for NADH binding also, although this residue is distinct from that at the catalytic site. The protection given by NADH against loss of activity is indicative of the close proximity of the active and NADH sites.

scholarly journals Photolabelling of Salmonella typhimurium LT2 sialidase. Identification of a peptide with a predicted structural similarity to the active sites of influenza-virus sialidases

1992 ◽  
Vol 285 (3) ◽  
pp. 957-964 ◽  
Author(s):  
T G Warner ◽  
R Harris ◽  
R McDowell ◽  
E R Vimr
Keyword(s):  
Salmonella Typhimurium ◽  
Active Site ◽  
Influenza A ◽  
Active Sites ◽  
Enzyme Inhibitors ◽  
Structural Similarity ◽  
Competitive Inhibitor ◽  
Beta Sheets ◽  
Sialidase Inhibitor ◽  
Active Site Cavity

The sialidase from Salmonella typhimurium LT2 was characterized by using photoaffinity-labelling techniques. The well-known sialidase inhibitor 5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non- 2-enonic acid (Neu5Ac2en) was modified to contain an amino group at C-9, which permitted the incorporation of 4-azidosalicylic acid in amide linkage at this position. Labelling of the purified protein with the radioactive (125I) photoprobe was determined to be highly specific for a region within the active-site cavity. This conclusion was based on the observation that the competitive inhibitor Neu5Ac2en in the photolysis mixture prevented labelling of the protein. In contrast, compounds with structural and chemical features similar to the probe and Neu5Ac2en, but which were not competitive enzyme inhibitors, did not affect the photolabelling of the protein. The peptide interacting with the probe was identified by CNBr treatment of the labelled protein, followed by N-terminal sequence analysis. Inspection of the primary structure of the protein, predicted from the cloned structural gene for the sialidase [Hoyer, Hamilton, Steenbergen & Vimr (1992) Mol. Microbiol. 6, 873-884] revealed that the label was incorporated into a 9.6 kDa fragment situated within the terminal third of the molecule near the C-terminal end. Secondary-structural predictions using the Garnier-Robson algorithm [Garnier, Osguthorpe & Robson (1978) J. Mol. Biol. 120, 97-120] of the labelled peptide revealed a structural similarity to the active site of influenza-A- and Sendai-HN-virus sialidases with a repetitive series of alternating beta-sheets connected with loops.

scholarly journals Relebactam Is a Potent Inhibitor of the KPC-2 β-Lactamase and Restores Imipenem Susceptibility in KPC-Producing Enterobacteriaceae

2018 ◽  
Vol 62 (6) ◽  
Author(s):  
Krisztina M. Papp-Wallace ◽  
Melissa D. Barnes ◽  
Jim Alsop ◽  
Magdalena A. Taracila ◽  
Christopher R. Bethel ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Rate Constant ◽  
Active Site ◽  
Klebsiella Oxytoca ◽  
Enterobacter Aerogenes ◽  
Clinical Isolates ◽  
Piperidine Ring ◽  
Water Molecules ◽  
Content Type ◽  
Site Water

ABSTRACT The imipenem-relebactam combination is in development as a potential treatment regimen for infections caused by Enterobacteriaceae possessing complex β-lactamase backgrounds. Relebactam is a β-lactamase inhibitor that possesses the diazabicyclooctane core, as in avibactam; however, the R1 side chain of relebactam also includes a piperidine ring, whereas that of avibactam is a carboxyamide. Here, we investigated the inactivation of the Klebsiella pneumoniae carbapenemase KPC-2, the most widespread class A carbapenemase, by relebactam and performed susceptibility testing with imipenem-relebactam using KPC-producing clinical isolates of Enterobacteriaceae . MIC measurements using agar dilution methods revealed that all 101 clinical isolates of KPC-producing Enterobacteriaceae ( K. pneumoniae , Klebsiella oxytoca , Enterobacter cloacae , Enterobacter aerogenes , Citrobacter freundii , Citrobacter koseri , and Escherichia coli ) were highly susceptible to imipenem-relebactam (MICs ≤ 2 mg/liter). Relebactam inhibited KPC-2 with a second-order onset of acylation rate constant ( k 2 / K ) value of 24,750 M −1 s −1 and demonstrated a slow off-rate constant ( k off ) of 0.0002 s −1 . Biochemical analysis using time-based mass spectrometry to map intermediates revealed that the KPC-2–relebactam acyl-enzyme complex was stable for up to 24 h. Importantly, desulfation of relebactam was not observed using mass spectrometry. Desulfation and subsequent deacylation have been observed during the reaction of KPC-2 with avibactam. Upon molecular dynamics simulations of relebactam in the KPC-2 active site, we found that the positioning of active-site water molecules is less favorable for desulfation in the KPC-2 active site than it is in the KPC-2–avibactam complex. In the acyl complexes, the water molecules are within 2.5 to 3 Å of the avibactam sulfate; however, they are more than 5 to 6 Å from the relebactam sulfate. As a result, we propose that the KPC-2–relebactam acyl complex is more stable than the KPC-2–avibactam complex. The clinical implications of this difference are not currently known.

scholarly journals Chemical Modification of the Active Site of the Acetylcholine Receptor

1969 ◽  
Vol 54 (1) ◽  
pp. 245-264 ◽  
Author(s):  
Arthur Karlin
Keyword(s):  
Chemical Modification ◽  
Acetylcholine Receptor ◽  
Disulfide Bond ◽  
Active Site ◽  
Quaternary Ammonium ◽  
Competitive Inhibitor ◽  
Affinity Labels ◽  
Electrophorus Electricus ◽  
Depolarizing Agents ◽  
Monoquaternary Ammonium

The receptor for acetylcholine in the subsynaptic membrane of the electroplax of Electrophorus electricus is a protein with a disulfide bond in the vicinity of the active site. This disulfide can be reduced and reoxidized with concomitant inhibition and restoration of the response to acetylcholine and other monoquaternary ammonium-depolarizing agents. Conversely, the bisquaternary hexamethonium, normally a competitive inhibitor, causes depolarization, and the activity of decamethonium is increased following reduction of the disulfide. The reduced receptor can be alkylated by various maleimide derivatives and is then no longer reoxidizable. Some quaternary ammonium maleimide derivatives act as affinity labels of the reduced receptor, alkylating it at a rate three orders of magnitude faster then do uncharged maleimide derivatives. Other types of potential affinity labels also react only with the reduced receptor and the resulting covalently attached quaternary ammonium moieties interact with the active site, strongly activating the receptor. These results suggest a model for the active site and its transitions in which an activator such as acetylcholine bridges between a negative subsite and a hydrophobic subsite in the vicinity of the disulfide, causing an altered conformation around the negative subsite and a decrasee of a few angstroms in the distance between the two subsites.

Competitive inhibitor binding to the binuclear copper active site in tyrosinase

1981 ◽  
Vol 103 (23) ◽  
pp. 7001-7003 ◽  
Author(s):  
Marjorie E. Winkler ◽  
Konrad Lerch ◽  
Edward I. Solomon
Keyword(s):  
Active Site ◽  
Competitive Inhibitor ◽  
Binuclear Copper ◽  
Inhibitor Binding

scholarly journals Acrolein, an irreversible active-site-directed inhibitor of deoxyribose 5-phosphate aldolase?

1976 ◽  
Vol 153 (2) ◽  
pp. 495-497 ◽  
Author(s):  
D C Wilton
Keyword(s):  
Schiff Base ◽  
Rate Constant ◽  
Active Site ◽  
Order Rate Constant ◽  
Lysine Residue ◽  
Prolonged Incubation ◽  
Enzyme Active Site ◽  
First Order ◽  
Substrate Analogue ◽  
Pseudo First Order

The enzyme deoxyribose 5-phosphate aldolase was irreversibly inactivated by the substrate analogue acrolein with a pseudo-first-order rate constant of 0.324 min-1 and a Ki (apparent) of 2.7 × 10(-4) m. No inactivation was observed after prolonged incubation with the epoxide analogues glycidol phosphate and glycidaldehyde. It is suggested that the acrolein is first activated by forming a Schiff base with the enzyme active-site lysine residue and it is the activated inhibitor that reacts with a suitable-active-site nucleophile.

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