scholarly journals Importance of the His-298 residue in the catalytic mechanism of the Streptomyces R61 extracellular dd-peptidase

1992 ◽  
Vol 282 (2) ◽  
pp. 495-500 ◽  
Author(s):  
A M Hadonou ◽  
M Jamin ◽  
M Adam ◽  
B Joris ◽  
J Dusart ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Marked Decrease ◽  
Cephalosporin C ◽  
Striking Result ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of ◽  
Substrate Hydrolysis

Among the active-site-serine penicillin-recognizing proteins, the Streptomyces R61 extracellular DD-peptidase is the only one to have a His-Thr-Gly sequence [instead of Lys-Thr(Ser)-Gly] in ‘box’ VII. The His residue was replaced by Gln or Lys. Both mutations induced a marked decrease in the rates of both tripeptide substrate hydrolysis and acylation by benzylpenicillin and cephalosporin C. The rate of hydrolysis of the thioester hippuryl thioglycollate was less affected. The most striking result was the disproportionate loss of transpeptidation properties by both mutants, indicating an important role of His-298 in this reaction. We believe that this result represents the first modification of a DD-peptidase leading to a specific decrease of the transpeptidation-to-hydrolysis ratio.

scholarly journals GlcNAcstatins are nanomolar inhibitors of human O-GlcNAcase inducing cellular hyper-O-GlcNAcylation

2009 ◽  
Vol 420 (2) ◽  
pp. 221-227 ◽  
Author(s):  
Helge C. Dorfmueller ◽  
Vladimir S. Borodkin ◽  
Marianne Schimpl ◽  
Daan M. F. van Aalten
Keyword(s):  
Active Site ◽  
Cell Biology ◽  
Rational Design ◽  
Catalytic Mechanism ◽  
Conserved Residues ◽  
Cellular Processes ◽  
Acetyl Group ◽  
Nanomolar Range ◽  
Insight Into

O-GlcNAcylation is an essential, dynamic and inducible post-translational glycosylation of cytosolic proteins in metazoa and can show interplay with protein phosphorylation. Inhibition of OGA (O-GlcNAcase), the enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, is a useful strategy to probe the role of this modification in a range of cellular processes. In the present study, we report the rational design and evaluation of GlcNAcstatins, a family of potent, competitive and selective inhibitors of human OGA. Kinetic experiments with recombinant human OGA reveal that the GlcNAcstatins are the most potent human OGA inhibitors reported to date, inhibiting the enzyme in the sub-nanomolar to nanomolar range. Modification of the GlcNAcstatin N-acetyl group leads to up to 160-fold selectivity against the human lysosomal hexosaminidases which employ a similar substrate-assisted catalytic mechanism. Mutagenesis studies in a bacterial OGA, guided by the structure of a GlcNAcstatin complex, provides insight into the role of conserved residues in the human OGA active site. GlcNAcstatins are cell-permeant and, at low nanomolar concentrations, effectively modulate intracellular O-GlcNAc levels through inhibition of OGA, in a range of human cell lines. Thus these compounds are potent selective tools to study the cell biology of O-GlcNAc.

scholarly journals The role of tyrosine-9 and the C-terminal helix in the catalytic mechanism of Alpha-class glutathione S-transferases

1999 ◽  
Vol 343 (3) ◽  
pp. 525-531 ◽  
Author(s):  
Claire S. ALLARDYCE ◽  
Paul D. MCDONAGH ◽  
Lu-Yun LIAN ◽  
C. Roland WOLF ◽  
Gordon C. K. ROBERTS
Keyword(s):  
Conformational Change ◽  
Catalytic Mechanism ◽  
Ethacrynic Acid ◽  
Marked Decrease ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Turnover Number ◽  
Hydrophobic Substrate ◽  
Glutathione S Transferases

Glutathione S-transferases (GSTs) play a key role in the metabolism of drugs and xenobiotics. To investigate the catalytic mechanism, substrate binding and catalysis by the wild-type and two mutants of GST A1-1 have been studied. Substitution of the ‘essential’ Tyr9 by phenylalanine leads to a marked decrease in the kcat for 1-chloro-2,4-dinitrobenzene (CDNB), but has no affect on kcat for ethacrynic acid. Similarly, removal of the C-terminal helix by truncation of the enzyme at residue 209 leads to a decrease in kcat for CDNB, but an increase in kcat for ethacrynic acid. The binding of a GSH analogue increases the affinity of the wild-type enzyme for CDNB, and increases the rate of the enzyme-catalysed conjugation of this substrate with the small thiols 2-mercaptoethanol and dithiothreitol. This suggests that GSH binding produces a conformational change which is transmitted to the binding site for the hydrophobic substrate, where it alters both the affinity for the substrate and the catalytic-centre activity (‘turnover number‘) for conjugation, perhaps by increasing the proportion of the substrate bound productively. Neither of these two effects of GSH analogues are seen in the C-terminally truncated enzyme, indicating a role for the C-terminal helix in the GSH-induced conformational change.

Investigation of the Catalytic Properties of the Dysthrombin, Thrombin Quick

1979 ◽  
Author(s):  
R Henriksen ◽  
W Owen ◽  
M Nesheim ◽  
K Mann
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Fluorescence Enhancement ◽  
Catalytic Mechanism ◽  
Antithrombin Iii ◽  
Catalytic Properties ◽  
Inhibitor Binding ◽  
Equivalent Number ◽  
Hydrolysis Of ◽  
Aggregation Of Platelets

Thrombin Quick (TQ) may be isolated following treatment of Prothrombin Quick [Owen, et al, Mayo Clinic Proceedings, 53: 29-33, (1978)] with Taipan venom, phospholipid and ca2+. The clotting activity of TQ with fibrinogen is 1/200 that of nornar thrombin (T). The activation of Factors V and VIII, and the aggregation of platelets by TQ occurs with an effectiveness of about 1/50 that of thrombin. when incubated with antithrombin III, both T ad TQ fom inhibitor complexes as determined by dodecylsulfate gel electropheresis. Titration of T and TQ with the fluorescent inhibitor dansylarginine-4-ethylpiperidine amide indicates an equivalent number of active sites based on protein absorption at 280 nm. However, the two enzymes may be distinquished by the decreased fluorescence enhancement observed with TQ relative to T, indicating an increased polarity in the inhibitor binding site of TQ. With the substrate benzoylarginine ethylester, TQ has a Km = 4.5 × 10-5M and kcat= 6.93 compared to Km = 4.0 × 10-5M and kcat= 17.7 for T. This indicates that the defect in TQ esterase activity is in the catalytic mechanism itself and not in substrate binding. The rate of inhibition of TQ by diisopropylphosphofluoridate is decreased. Decreased acylation and deacylation rates for TQ relative to T are observed for hydrolysis of the active site titrant 4-methykl-umbelliferyl-p-guanidinobenzoate

scholarly journals Structural Characterization of an S-enantioselective Imine Reductase from Mycobacterium Smegmatis

Biomolecules ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 1130
Author(s):  
Timo Meyer ◽  
Nadine Zumbrägel ◽  
Christina Geerds ◽  
Harald Gröger ◽  
Hartmut H. Niemann
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Mycobacterium Smegmatis ◽  
Catalytic Mechanism ◽  
Building Blocks ◽  
Secondary Amines ◽  
Hydrophobic Amino Acids ◽  
High Degree

NADPH-dependent imine reductases (IREDs) are enzymes capable of enantioselectively reducing imines to chiral secondary amines, which represent important building blocks in the chemical and pharmaceutical industry. Since their discovery in 2011, many previously unknown IREDs have been identified, biochemically and structurally characterized and categorized into families. However, the catalytic mechanism and guiding principles for substrate specificity and stereoselectivity remain disputed. Herein, we describe the crystal structure of S-IRED-Ms from Mycobacterium smegmatis together with its cofactor NADPH. S-IRED-Ms belongs to the S-enantioselective superfamily 3 (SFam3) and is the first IRED from SFam3 to be structurally described. The data presented provide further evidence for the overall high degree of structural conservation between different IREDs of various superfamilies. We discuss the role of Asp170 in catalysis and the importance of hydrophobic amino acids in the active site for stereospecificity. Moreover, a separate entrance to the active site, potentially functioning according to a gatekeeping mechanism regulating access and, therefore, substrate specificity is described.

The polysulphate binding domain of human proacrosin/acrosin is involved in both the enzyme activation and spermatozoa-zona pellucida interaction

Zygote ◽  
1998 ◽  
Vol 6 (1) ◽  
pp. 75-83 ◽  
Author(s):  
R. D. Moreno ◽  
M. S. Sepúlveda ◽  
A. de Ioannes ◽  
C. Barros
Keyword(s):  
Zona Pellucida ◽  
Active Site ◽  
Enzyme Activation ◽  
Binding Domain ◽  
Present Evidence ◽  
Sperm Binding ◽  
Mammalian Sperm ◽  
Hydrolysis Of

SummaryMammalian acrosin is a protease present as a zymogen in the acrosome of a non-reacted mammalian sperm, and in vitro is able to carry out limited hydrolysis of homologous and heterologous zonae pellucidae. On the other hand, sulphated polymers and zona pellcida glycoproteins bind to acrosin on a domain different from the active site, named the polysulphate binding domain (PSBD). Thus it is believed that acrosome-reacted spermatozoa bind to glycan chains of the zona pellucida through PSBD participating as secondary binding receptor. The aim of the present work was to study the role of PSBD during both human gamete interaction and acrosin activation. In this work we present evidence that the anti-human acrosin monoclonal antibody C5F10 is directed to an epitope located on or near the PSBD on human proacrosin/acrosin. Moreover, we show that this antibody is able to inhibit both proacrosin activation induced by fucoidan and the sperm binding to the zona pellucida. Our results suggest that the same PSBD is involved in both sperm secondary binding, during zona pellucida penetration, and proacrosin activation.

Characteristics of Aerobacter beta-lactamase

1968 ◽  
Vol 14 (2) ◽  
pp. 139-145 ◽  
Author(s):  
M. Goldner ◽  
D. G. Glass ◽  
P. C. Fleming
Keyword(s):  
Reaction Rates ◽  
Penicillin G ◽  
Cephalosporin C ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Lactam Ring ◽  
Rate Of Hydrolysis ◽  
High Concentrations ◽  
Hydrolysis Of ◽  
Pseudomonas Pyocyanea

In this investigation, Aerobacter cloacae is shown to inactivate cephalosporin by hydrolysis of its beta-lactam ring. This was demonstrated by iodine absorption and infrared absorption spectra.The values of the Michaelis constant obtained with cephalosporin C and deacetyl cephalosporin C indicate a great affinity of the Aerobacter's beta-lactamase for its substrate. The enzyme was most active at pH 7.0 and 37 C. Aqueous washings of the Aerobacter cells were a potent source of enzyme.The beta-lactamase of A. cloacae was active on both cephalosporin and penicillin. A higher rate of hydrolysis was observed with cephalosporin C and deacetyl cephalosporin C than with cephalothin and cephaloridine. The ratio of reaction rates on cephalosporin C to that on penicillin G was consistently of the order of 100 to 1. The activity on V, N, and especially the semisynthetic penicillins was also low.The A. cloacae enzyme was easily demonstrable in large amount without added inducer. By contrast, the activity of the beta-lactamase from Pseudomonas pyocyanea cannot be detected unless high concentrations of inducer are used.

scholarly journals KPC-2 β-lactamase Enables Carbapenem Antibiotic Resistance Through Fast Deacylation of the Covalent Intermediate

2020 ◽  
pp. jbc.RA120.015050
Author(s):  
Shrenik C Mehta ◽  
Ian M Furey ◽  
Orville A Pemberton ◽  
David M Boragine ◽  
Yu Chen ◽  
...  
Keyword(s):  
Active Site ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
The Common ◽  
Covalent Intermediate ◽  
Hydrolysis Of ◽  
Carbapenem Antibiotic ◽  
Enzyme Intermediate

Serine active-site β-lactamases hydrolyze β-lactam antibiotics through formation of a covalent acyl-enzyme intermediate followed by deacylation via an activated water molecule. Carbapenem antibiotics are poorly hydrolyzed by most β-lactamases due to slow hydrolysis of the acyl-enzyme intermediate. However, the emergence of the KPC-2 carbapenemase has resulted in widespread resistance to these drugs, suggesting it operates more efficiently. Here, we investigated the unusual features of KPC-2 that enable this resistance. We show that KPC-2 has a 20,000-fold increased deacylation rate compared to the common TEM-1 β-lactamase. Further, kinetic analysis of active site alanine mutants indicates that carbapenem hydrolysis is a concerted effort involving multiple residues. Substitution of Asn170 greatly decreases the deacylation rate, but this residue is conserved in both KPC-2 and non-carbapenemase β-lactamases, suggesting it promotes carbapenem hydrolysis only in the context of KPC-2. X-ray structure determination of the N170A enzyme in complex with hydrolyzed imipenem suggests Asn170 may prevent the inactivation of the deacylating water by the 6α-hydroxyethyl substituent of carbapenems. In addition, the Thr235 residue, which interacts with the C3 carboxylate of carbapenems, also contributes strongly to the deacylation reaction. In contrast, mutation of the Arg220 and Thr237 residues decreases the acylation rate and, paradoxically, improves binding affinity for carbapenems. Thus, the role of these residues may be ground state destabilization of the enzyme-substrate complex or, alternatively, to ensure proper alignment of the substrate with key catalytic residues to facilitate acylation. These findings suggest modifications of the carbapenem scaffold to avoid hydrolysis by KPC-2 β-lactamase.

An ATP-inhibited soluble 5'-nucleotidase of rat kidney

1988 ◽  
Vol 254 (2) ◽  
pp. F191-F195
Author(s):  
M. Le Hir ◽  
U. C. Dubach
Keyword(s):  
High Speed ◽  
Catalytic Properties ◽  
Divalent Cations ◽  
Rat Kidney ◽  
Maximal Activity ◽  
Rate Of Hydrolysis ◽  
Membrane Bound ◽  
Ph Range ◽  
Hydrolysis Of

Hydrolysis of 5'-AMP by 5'-nucleotidase is a possible source of adenosine in the kidney. A renal membrane-bound ecto-5'-nucleotidase has been previously described. The present study deals with the catalytic properties of a 5'-AMP phosphohydrolase partially purified from high-speed supernatants of rat kidney homogenates. It exhibits phosphatase activity toward 5'-AMP, 5'-IMP, and 5'-GMP, but not toward 2'- and 3'-AMP and corresponds therefore to a 5'-nucleotidase. The hydrolysis of 5'-AMP by the soluble 5'-nucleotidase requires divalent cations. Maximal activity is reached with 10 microM of either Mn2+ or Co2+, whereas half-maximal activity is obtained with approximately 400 microM Mg2+. The soluble 5'-nucleotidase exhibits Michaelis-Menten kinetics with a Km of 9.5 microM for 5'-AMP. In the presence of 1 mM of free Mg2+, physiological concentrations of ATP provoke an increase of the Km for 5'-AMP and a decrease of Vmax. An increase of the pH of 0.4 units in the pH range 6.4-7.4 roughly doubles the rate of hydrolysis of 5'-AMP. The effects of ATP and of the pH are compatible with a role of the renal soluble 5'-nucleotidase in the hydrolysis of 5'-AMP and in the production of adenosine during hypoxia.

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