scholarly journals Substrate specificity and kinetic properties of α-galactosidases from Vicia faba

1969 ◽  
Vol 115 (1) ◽  
pp. 47-54 ◽  
Author(s):  
P. M. Dey ◽  
J. B. Pridham
Keyword(s):  
Catalytic Activity ◽  
Substrate Specificity ◽  
Kinetic Parameters ◽  
Vicia Faba ◽  
Active Site ◽  
Effect Of Temperature ◽  
Kinetic Properties ◽  
Photo Oxidation ◽  
Hydrolysis Of ◽  
Enzyme I

1. The hydrolysis of a variety of galactosides and other glycosides by α-galactosidases I and II of Vicia faba was studied. 2. The effect of temperature on kinetic parameters was also examined. 3. Both enzymes are inhibited by excess of substrate (p-nitrophenyl α-d-galactoside); with enzyme I this is competitive and is caused by the galactosyl moiety. 4. Enzyme I is inhibited by oligosaccharides possessing terminal non-reducing galactose residues and to a smaller extent by l-arabinose and d-fucose. 5. The effect of pH on Km and Vmax. values suggests that carboxyl and imidazole groups are involved in the catalytic activity of enzyme I. 6. Photo-oxidation experiments with enzyme I also suggest that an imidazole group is present at the active site.

Length of the active-site crossover loop defines the substrate specificity of ubiquitin C-terminal hydrolases for ubiquitin chains

2011 ◽  
Vol 441 (1) ◽  
pp. 143-149 ◽  
Author(s):  
Zi-Ren Zhou ◽  
Yu-Hang Zhang ◽  
Shuai Liu ◽  
Ai-Xin Song ◽  
Hong-Yu Hu
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Dependent Manner ◽  
Deubiquitinating Enzymes ◽  
Chain Flexibility ◽  
Isopeptide Bond ◽  
Short Loop ◽  
The One ◽  
Isopeptidase Activity ◽  
Hydrolysis Of

UCHs [Ub (ubiquitin) C-terminal hydrolases] are a family of deubiquitinating enzymes that are often thought to only remove small C-terminal peptide tails from Ub adducts. Among the four UCHs identified to date, neither UCH-L3 nor UCH-L1 can catalyse the hydrolysis of isopeptide Ub chains, but UCH-L5 can when it is present in the PA700 complex of the proteasome. In the present paper, we report that the UCH domain of UCH-L5, different from UCH-L1 and UCH-L3, by itself can process the K48-diUb (Lys48-linked di-ubiquitin) substrate by cleaving the isopeptide bond between two Ub units. The catalytic specificity of the four UCHs is dependent on the length of the active-site crossover loop. The UCH domain with a long crossover loop (usually >14 residues), such as that of UCH-L5 or BAP1 [BRCA1 (breast cancer early-onset 1)-associated protein 1], is able to cleave both small and large Ub derivatives, whereas the one with a short loop can only process small Ub derivatives. We also found that elongation of the crossover loop enables UCH-L1 to have isopeptidase activity for K48-diUb in a length-dependent manner. Thus the loop length of UCHs defines their substrate specificity for diUb chains, suggesting that the chain flexibility of the crossover loop plays an important role in determining its catalytic activity and substrate specificity for cleaving isopeptide Ub chains.

scholarly journals Amino Acid Residues That Contribute to Substrate Specificity of Class A β-Lactamase SME-1

2005 ◽  
Vol 49 (8) ◽  
pp. 3421-3427 ◽  
Author(s):  
Fahd K. Majiduddin ◽  
Timothy Palzkill
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Class A ◽  
Study Support ◽  
Functional Mutants ◽  
Hydrolysis Of ◽  
Traditional Class

ABSTRACT Carbapenem antibiotics are used as antibiotics of last resort because they possess a broad spectrum of antimicrobial activity and are not easily hydrolyzed by β-lactamases. Recently, class A enzymes, such as the SME-1, NMC-A, and IMI-1 β-lactamases, have been identified with the capacity to hydrolyze carbapenem antibiotics. Traditional class A β-lactamases, such as TEM-1 and SHV-1, are unable to hydrolyze carbapenem antibiotics and exhibit some differences in sequence from those that are able to hydrolyze carbapenem antibiotics. The positions that differ may contribute to the unique substrate specificity of the class A carbapenemase SME-1. Codons in the SME-1 gene representing residues 104, 105, 132, 167, 237, and 241 were randomized by site-directed mutagenesis, and functional mutants were selected for the ability to hydrolyze imipenem, ampicillin, or cefotaxime. Although several positions are important for hydrolysis of β-lactam antibiotics, no single position was found to uniquely contribute to carbapenem hydrolysis. The results of this study support a model whereby the carbapenemase activity of SME-1 is due to a highly distributed set of interactions that subtly alter the structure of the active-site pocket.

scholarly journals Mechanism of the reaction catalysed by cytosolic 5′-nucleotidase/phosphotransferase: formation of a phosphorylated intermediate

1996 ◽  
Vol 317 (3) ◽  
pp. 797-801 ◽  
Author(s):  
Cristina BAIOCCHI ◽  
Rossana PESI ◽  
Marcella CAMICI ◽  
Roichi ITOH ◽  
Maria GRAZIA TOZZI
Keyword(s):  
Reaction Mechanism ◽  
Kinetic Parameters ◽  
Acid Treatment ◽  
Histidine Residue ◽  
Thiol Oxidation ◽  
Photo Oxidation ◽  
Calf Thymus ◽  
Hydrolysis Of ◽  
Mechanism Of The Reaction

Cytosolic 5´-nucleotidase preferentially catalysing the hydrolysis of IMP, GMP and their deoxy derivatives, and endowed with phosphotransferase activity, was purified from calf thymus and its reaction mechanism was studied. In the presence of [32P]IMP, ATP and MgCl2, a covalent enzyme–phosphate intermediate was trapped by mixing with an SDS solution. Heat or acid treatment of the enzyme before incubation with radiolabelled substrate prevented formation of the intermediate. Furthermore, on the basis of studies on the kinetic parameters of the enzyme as function of pH, and of experiments on thiol oxidation and photo-oxidation, we suggest the involvement of cysteine and histidine residue(s) in the reaction mechanism.

scholarly journals Kinetic studies on glucoamylase of rabbit small intestine

1976 ◽  
Vol 153 (2) ◽  
pp. 321-327 ◽  
Author(s):  
S Sivakami ◽  
A N Radhakrishnan
Keyword(s):  
Small Intestine ◽  
Substrate Specificity ◽  
Kinetic Studies ◽  
Kinetic Properties ◽  
Ph Optimum ◽  
Kinetics Of Hydrolysis ◽  
Brush Borders ◽  
Rabbit Small Intestine ◽  
Kinetics Of ◽  
Hydrolysis Of

The kinetic properties of a maltase-glucoamylase complex with a neutral pH optimum, purified to homogeneity from the brush borders of the rabbit small intestine, are described. It has a broad range of substrate specificity, hydrolysing di- and poly-saccharides with α-1,4 and α-1,6 linkages. The Km and Vmax, values of the enzyme for the various substrates were determined. Starch and maltose were its best substrates. The kinetics of hydrolysis of two synthetic linear maltosaccharides, namely maltotriose and maltopentaose, were studied. Mixed-substrate incubation studies revealed the presence of at least two interacting sites on the enzyme, and the data were further analysed by the use of a number of non-substrate inhibitors.

scholarly journals Antagonism between substitutions in β-lactamase explains a path not taken in the evolution of bacterial drug resistance

2020 ◽  
Vol 295 (21) ◽  
pp. 7376-7390
Author(s):  
Cameron A. Brown ◽  
Liya Hu ◽  
Zhizeng Sun ◽  
Meha P. Patel ◽  
Sukrit Singh ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Active Site ◽  
X Ray ◽  
X Ray Crystallography ◽  
Naturally Occurring ◽  
Bacterial Drug Resistance ◽  
Evolutionary Trajectories ◽  
Dynamics Simulations ◽  
Negative Epistasis ◽  
Hydrolysis Of

CTX-M β-lactamases are widespread in Gram-negative bacterial pathogens and provide resistance to the cephalosporin cefotaxime but not to the related antibiotic ceftazidime. Nevertheless, variants have emerged that confer resistance to ceftazidime. Two natural mutations, causing P167S and D240G substitutions in the CTX-M enzyme, result in 10-fold increased hydrolysis of ceftazidime. Although the combination of these mutations would be predicted to increase ceftazidime hydrolysis further, the P167S/D240G combination has not been observed in a naturally occurring CTX-M variant. Here, using recombinantly expressed enzymes, minimum inhibitory concentration measurements, steady-state enzyme kinetics, and X-ray crystallography, we show that the P167S/D240G double mutant enzyme exhibits decreased ceftazidime hydrolysis, lower thermostability, and decreased protein expression levels compared with each of the single mutants, indicating negative epistasis. X-ray structures of mutant enzymes with covalently trapped ceftazidime suggested that a change of an active-site Ω-loop to an open conformation accommodates ceftazidime leading to enhanced catalysis. 10-μs molecular dynamics simulations further correlated Ω-loop opening with catalytic activity. We observed that the WT and P167S/D240G variant with acylated ceftazidime both favor a closed conformation not conducive for catalysis. In contrast, the single substitutions dramatically increased the probability of open conformations. We conclude that the antagonism is due to restricting the conformation of the Ω-loop. These results reveal the importance of conformational heterogeneity of active-site loops in controlling catalytic activity and directing evolutionary trajectories.

scholarly journals Multiple substitutions at position 104 of β-lactamase TEM-1: assessing the role of this residue in substrate specificity

1995 ◽  
Vol 305 (1) ◽  
pp. 33-40 ◽  
Author(s):  
A Petit ◽  
L Maveyraud ◽  
F Lenfant ◽  
J P Samama ◽  
R Labia ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Kinetic Properties ◽  
Third Generation ◽  
Wild Type Enzyme ◽  
Beta Lactamases ◽  
Class A ◽  
Extended Spectrum ◽  
Third Generation Cephalosporins

Residue 104 is frequently mutated from a glutamic acid to a lysine in the extended-spectrum TEM beta-lactamases responsible for the resistance to third-generation cephalosporins in clinical Gram negative strains. Among class A beta-lactamases, it is the most variable residue within a highly conserved loop which delineates one side of the active site of the enzymes. To investigate the role of this residue in the extended-spectrum phenotype, it has been replaced by serine, threonine, lysine, arginine, tyrosine and proline. All these substitutions yield active enzymes, with no drastic changes in kinetic properties compared with the wild-type enzyme, except with cefaclor, but an overall improved affinity for second- and third-generation cephalosporins. Only mutant E104K exhibits a significant ability to hydrolyse cefotaxime. Molecular modelling shows that the substitutions have generally no impact on the conformation of the 101-111 loop as the side chains of residues at position 104 are all turned towards the solvent. Unexpectedly, the E104P mutant turns out to be the most efficient enzyme. All our results argue in favour of an indirect role for this residue 104 in the substrate specificity of the class A beta-lactamases. This residue contributes to the precise positioning of residues 130-132 which are involved in substrate binding and catalysis. Changing residue 104 could also modify slightly the local electrostatic potential in this part of the active site. The limited kinetic impact of the mutations at this position have to be analysed in the context of the microbiological problem of resistance to third-generation cephalosporins. Although mutation E104K improves the ability of the enzyme to hydrolyse these compounds, it is not sufficient to confer true resistance, and is always found in clinical isolates associated with at least one mutation at another part of the active site. It is the combined effect of the two mutations that synergistically enhances the hydrolytic capability of the enzyme towards third-generation cephalosporins.

scholarly journals A general method to predict the effect of single amino acid substitutions on enzyme catalytic activity

2017 ◽  
Author(s):  
Yu-Hsiu T. Lin ◽  
Cheng Lai Victor Huang ◽  
Christina Ho ◽  
Max Shatsky ◽  
Jack F. Kirsch
Keyword(s):  
Amino Acid ◽  
Kinetic Parameters ◽  
Active Site ◽  
Enzyme Reaction ◽  
Site Directed Mutagenesis ◽  
Enzyme Function ◽  
Single Amino Acid ◽  
Kinetic Properties ◽  
Amino Acid Substitutions ◽  
Active Site Residues

ABSTRACTOver the past thirty years, site-directed mutagenesis has become established as one of the most powerful techniques to probe enzyme reaction mechanisms1-3. Substitutions of active site residues are most likely to yield significant perturbations in kinetic parameters, but there are many examples of profound changes in these values elicited by remote mutations4-6. Ortholog comparisons of extant sequences show that many mutations do not have profound influence on enzyme function. As the number of potential single natural amino acid substitutions that can be introduced in a protein of N amino acids in length by directed mutation is very large (19 * N), it would be useful to have a method to predict which amino acid substitutions are more likely to introduce significant changes in kinetic parameters in order to design meaningful probes into enzyme function. What is especially desirable is the identification of critical residues that do not contact the substrate directly, and may be remote from the active site.We collected literature data reflecting the effects of 2,804 mutations on kinetic properties for 12 enzymes. These data along with characteristic predictors were used in a machine-learning scheme to train a classifier to predict the effect of mutation. Use of this algorithm allows one to predict with a 2.5-fold increase in precision, if a given mutation, made anywhere in the enzyme, will cause a decrease in kcat/Km value of ≥ 95%. The improved precision allows the experimentalist to reduce the number of mutations necessary to probe the enzyme reaction mechanism.

scholarly journals The molecular structure of Schistosoma mansoni PNP isoform 2 provides insights into the nucleotide selectivity of PNPs

2018 ◽  
Author(s):  
Juliana Roberta Torini ◽  
Larissa Romanello ◽  
Fernanda Aparecida Heleno Batista ◽  
Vitor Hugo Balasco Serrão ◽  
Muhammad Faheem ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Schistosoma Mansoni ◽  
Binding Site ◽  
Kinetic Parameters ◽  
Isothermal Titration Calorimetry ◽  
Active Site ◽  
Binding Mode ◽  
Titration Calorimetry ◽  
Purine Salvage ◽  
Key Enzyme

AbstractPurine nucleoside phosphorylases (PNPs) play an important role in the blood fluke parasite Schistosoma mansoni as a key enzyme of the purine salvage pathway. Here we present the structural and kinetic characterization of a new PNP isoform from S. mansoni, named as SmPNP2. Screening of different ligands using a thermofluorescence approach indicated cytidine and cytosine as potential ligands. The binding of cytosine was confirmed by isothermal titration calorimetry, with a KD of 27 μM, and kinetic parameters for cytidine catalysis were obtained by ITC resulting in a KM of 76.3 μM. SmPNP2 also displays catalytic activity against inosine and adenosine, making it the first described PNP with robust catalytic activity towards both pyrimidines and purines. Crystallographic structures of SmPNP2 with different ligands were obtained and comparison of these structures with the previously described S. mansoni PNP (SmPNP1) provided clues for the unique capability of SmPNP2 to bind pyrimidines. When compared with the structure of SmPNP1, substitutions in the vicinity of SmPNP2 active site alter the architecture of the nucleoside base binding site allowing an alternative binding mode for nucleosides, with a 180° rotation from the canonical binding mode. The remarkable plasticity of this binding site deepens the understanding of the correlation between structure and nucleotide selectivity, offering new ways to analyses PNP activity.Author SummarySchistosoma mansoni is a human parasite dependent on purine salvage for purine bases supply. Purine nucleoside phosphorylase (PNP) is a key enzyme in this pathway. It carries two PNP isoforms, one previously characterized (SmPNP1) and one unknown (SmPNP2). Here we present the crystallographic structure of SmPNP2 and its complex with cytosine, cytidine, ribose-l-phosphate, adenine, hypoxanthine, and tubercidin. Cytidine and cytosine were identified as ligands of SmPNP2 using a thermofluorescence approach. Binding of cytosine was proven by Isothermal Titration Calorimetry (ITC) and cytidine, inosine, and adenosine kinetic parameters were also obtained. Purine bases showed different binding in the active site, rotated 180° from the canonical binding mode. It’s the first report showing a Low Molecular Mass PNP capable of catalyzing both types of nucleotide bases. The SmPNP2 odd behavior sheds a new light on the Schistosoma mansoni’s life cycle metabolic adaptation.

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