scholarly journals Evidence for ‘lock and key’ character in an anti-phosphonate hydrolytic antibody catalytic site augmented by non-reaction centre recognition: variation in substrate selectivity between an anti-phosphonate antibody, an anti-phosphate antibody and two hydrolytic enzymes

2004 ◽  
Vol 381 (1) ◽  
pp. 125-130 ◽  
Author(s):  
Sanjiv SONKARIA ◽  
Guillaume BOUCHER ◽  
José FLÓREZ-ÁLVAREZ ◽  
Bilal SAID ◽  
Syeed HUSSAIN ◽  
...  
Keyword(s):  
Hydrolytic Enzymes ◽  
Catalytic Site ◽  
Catalytic Antibody ◽  
Substrate Selectivity ◽  
Reaction Centre ◽  
Antibody Preparation ◽  
Leaving Group ◽  
Catalytically Active ◽  
Group Part ◽  
Kinetic Selectivity

The substrate selectivities of an anti-phosphonate and an anti-phosphate kinetically homogeneous polyclonal catalytic antibody preparation and two hydrolytic enzymes were compared by using hapten-analogous and truncated carbonate and ester substrates each containing a 4-nitrophenolate leaving group. Syntheses of the truncated substrates devoid of recognition features in the non-leaving group parts of the substrates are reported. The relatively high kinetic selectivity of the more active anti-phosphonate antibody preparation is considered to depend on a relatively rigid catalytic site with substantial reaction centre specificity together with other important recognition interactions with the extended non-leaving group part of the substrate. In contrast, the less catalytically active, more flexible anti-phosphate antibody exhibits much lower kinetic selectivity for the substrate reaction centre comparable with that of the hydrolytic enzymes with activity much less dependent on recognition interactions with the non-leaving group part of the substrate. The ways in which haptenic flexibility and IgG architecture might contribute to the differential kinetic selectivities are indicated.

scholarly journals Improvement in hydrolytic antibody activity by change in haptenic structure from phosphate to phosphonate with retention of a common leaving-group determinant: evidence for the ‘flexibility’ hypothesis

2003 ◽  
Vol 376 (3) ◽  
pp. 813-821 ◽  
Author(s):  
Sheraz GUL ◽  
Sanjiv SONKARIA ◽  
Surapong PINITGLANG ◽  
José FLOREZ-ALVAREZ ◽  
Syeed HUSSAIN ◽  
...  
Keyword(s):  
Structural Motif ◽  
Catalytic Antibody ◽  
Antibody Activity ◽  
Reaction Centre ◽  
Binding Characteristics ◽  
Leaving Group ◽  
Catalytic Mechanisms ◽  
Leaving Groups ◽  
Antibody Preparations ◽  
Ester Substrate

To investigate the hypothesis that decreased hapten flexibility may lead to increased catalytic antibody activity, we used two closely related immunogens differing only in the flexibility of the atomic framework around the structural motif of the haptens, analogous to the reaction centre of the corresponding substrates. Identical leaving-group determinants in the haptens and identical leaving groups in the substrates removed the ambiguity inherent in some data reported in the literature. Anti-phosphate and anti-phosphonate kinetically homogeneous polyclonal catalytic antibody preparations were compared by using carbonate and ester substrates respectively, each containing a 4-nitrophenolate leaving group. Synthetic routes to a new phosphonate hapten and new ester substrate were developed. The kinetic advantage of the more rigid anti-phosphonate/ester system was demonstrated at pH 8.0 by a 13-fold advantage in kcat/knon-cat and a 100-fold advantage in the proficiency constant, kcat/knon-cat·Km. Despite these differences, the pH-dependences of the kinetic and binding characteristics and the results of chemical modification studies suggest closely similar catalytic mechanisms. The possible origin of the kinetic advantage of the more rigid hapten/substrate system is discussed.

Determination of the catalytic site content of a polyclonal catalytic antibody preparation

1998 ◽  
Vol 26 (2) ◽  
pp. S170-S170 ◽  
Author(s):  
MARINA RESMINI ◽  
SHERAZ GUL ◽  
SANJIV SONKARIA ◽  
GERARD GALLACHER ◽  
KEITH BROCKLEHURST
Keyword(s):  
Catalytic Site ◽  
Catalytic Antibody ◽  
Antibody Preparation

scholarly journals Evidence that the mechanism of antibody-catalysed hydrolysis of arylcarbamates can be determined by the structure of the immunogen used to elicit the catalytic antibody

2007 ◽  
Vol 401 (3) ◽  
pp. 721-726 ◽  
Author(s):  
Guillaume Boucher ◽  
Bilal Said ◽  
Elizabeth L. Ostler ◽  
Marina Resmini ◽  
Keith Brocklehurst ◽  
...  
Keyword(s):  
Phenyl Group ◽  
Catalytic Antibody ◽  
Antibody Preparation ◽  
Tetrahedral Intermediate ◽  
Rate Determining Step ◽  
Catalysed Reaction ◽  
Leaving Group ◽  
Antibody Catalysis ◽  
Elicitation Process ◽  
Hydrolysis Of

A kinetically homogeneous anti-phosphate catalytic antibody preparation was shown to catalyse the hydrolysis of a series of O-aryl N-methyl carbamates containing various substituents in the 4-position of the O-phenyl group. The specific nature of the antibody catalysis was demonstrated by the adherence of these reactions to the Michaelis–Menten equation, the complete inhibition by a hapten analogue, and the failure of the antibody to catalyse the hydrolysis of the 2-nitrophenyl analogue of the 4-nitrophenylcarbamate substrate. Hammett σ–ρ analysis suggests that both the non-catalysed and antibody-catalysed reactions proceed by mechanisms in which development of the aryloxyanion of the leaving group is well advanced in the transition state of the rate-determining step. This is probably the ElcB (elimination–addition) mechanism for the non-catalysed reaction, but for the antibody-catalysed reaction might be either ElcB or BAc2 (addition–elimination), in which the elimination of the aryloxy group from the tetrahedral intermediate has become rate-determining. This result provides evidence of the dominance of recognition of phenolate ion character in the phosphate hapten in the elicitation process, and is discussed in connection with data from the literature that suggest a BAc2 mechanism, with rate-determining formation of the tetrahedral intermediate for the hydrolysis of carbamate substrates catalysed by an antibody elicited by a phosphonamidate hapten in which phenolate anion character is minimized. The present paper contributes to the growing awareness that small differences in the structure of haptens can produce large differences in catalytic characteristics.

scholarly journals Inverse enzyme isotope effects in human purine nucleoside phosphorylase with heavy asparagine labels

2018 ◽  
Vol 115 (27) ◽  
pp. E6209-E6216 ◽  
Author(s):  
Rajesh K. Harijan ◽  
Ioanna Zoi ◽  
Dimitri Antoniou ◽  
Steven D. Schwartz ◽  
Vern L. Schramm
Keyword(s):  
Transition State ◽  
Purine Nucleoside Phosphorylase ◽  
Isotope Effects ◽  
Catalytic Site ◽  
Path Sampling ◽  
Transition Path ◽  
Transition Path Sampling ◽  
Steady State Kinetics ◽  
Leaving Group ◽  
Transition State Barrier

Transition path-sampling calculations with several enzymes have indicated that local catalytic site femtosecond motions are linked to transition state barrier crossing. Experimentally, femtosecond motions can be perturbed by labeling the protein with amino acids containing 13C, 15N, and nonexchangeable 2H. A slowed chemical step at the catalytic site with variable effects on steady-state kinetics is usually observed for heavy enzymes. Heavy human purine nucleoside phosphorylase (PNP) is slowed significantly (kchemlight/kchemheavy = 1.36). An asparagine (Asn243) at the catalytic site is involved in purine leaving-group activation in the PNP catalytic mechanism. In a PNP produced with isotopically heavy asparagines, the chemical step is faster (kchemlight/kchemheavy = 0.78). When all amino acids in PNP are heavy except for the asparagines, the chemical step is also faster (kchemlight/kchemheavy = 0.71). Substrate-trapping experiments provided independent confirmation of improved catalysis in these constructs. Transition path-sampling analysis of these partially labeled PNPs indicate altered femtosecond catalytic site motions with improved Asn243 interactions to the purine leaving group. Altered transition state barrier recrossing has been proposed as an explanation for heavy-PNP isotope effects but is incompatible with these isotope effects. Rate-limiting product release governs steady-state kinetics in this enzyme, and kinetic constants were unaffected in the labeled PNPs. The study suggests that mass-constrained femtosecond motions at the catalytic site of PNP can improve transition state barrier crossing by more frequent sampling of essential catalytic site contacts.

scholarly journals Activation and selectivity of OTUB-1 and OTUB-2 deubiquitinylases

2020 ◽  
Vol 295 (20) ◽  
pp. 6972-6982
Author(s):  
Dakshinamurthy Sivakumar ◽  
Vikash Kumar ◽  
Michael Naumann ◽  
Matthias Stein
Keyword(s):  
Active Site ◽  
Electrostatic Interactions ◽  
Active Form ◽  
Cysteine Proteases ◽  
Binding Pocket ◽  
Catalytic Triad ◽  
Catalytic Site ◽  
Dynamics Simulation ◽  
Active Site Residues ◽  
Catalytically Active

The ovarian tumor domain (OTU) deubiquitinylating cysteine proteases OTUB1 and OTUB2 (OTU ubiquitin aldehyde binding 1 and 2) are representative members of the OTU subfamily of deubiquitinylases. Deubiquitinylation critically regulates a multitude of important cellular processes, such as apoptosis, cell signaling, and growth. Moreover, elevated OTUB expression has been observed in various cancers, including glioma, endometrial cancer, ovarian cancer, and breast cancer. Here, using molecular dynamics simulation approaches, we found that both OTUB1 and OTUB2 display a catalytic triad characteristic of proteases but differ in their configuration and protonation states. The OTUB1 protein had a prearranged catalytic site, with strong electrostatic interactions between the active-site residues His265 and Asp267. In OTUB2, however, the arrangement of the catalytic triad was different. In the absence of ubiquitin, the neutral states of the catalytic-site residues in OTUB2 were more stable, resulting in larger distances between these residues. Only upon ubiquitin binding did the catalytic triad in OTUB2 rearrange and bring the active site into a catalytically feasible state. An analysis of water access channels revealed only a few diffusion trajectories for the catalytically active form of OTUB1, whereas in OTUB2 the catalytic site was solvent-accessible, and a larger number of water molecules reached and left the binding pocket. Interestingly, in OTUB2, the catalytic residues His224 and Asn226 formed a stable hydrogen bond. We propose that the observed differences in activation kinetics, protonation states, water channels, and active-site accessibility between OTUB1 and OTUB2 may be relevant for the selective design of OTU inhibitors.

scholarly journals Cloning and characterization of ADAM28: evidence for autocatalytic pro-domain removal and for cell surface localization of mature ADAM28

2000 ◽  
Vol 348 (1) ◽  
pp. 21-27 ◽  
Author(s):  
Linda HOWARD ◽  
Rose A. MACIEWICZ ◽  
Carl P. BLOBEL
Keyword(s):  
Cell Surface ◽  
Secretory Pathway ◽  
Active Form ◽  
Catalytic Site ◽  
Mouse Lung ◽  
Mutant Form ◽  
Sequence Comparisons ◽  
Mouse Tissues ◽  
Catalytically Active ◽  
Alternatively Spliced

The metalloprotease disintegrins are a family of membrane-anchored glycoproteins with diverse functions in fertilization, myoblast fusion, neurogenesis and protein ectodomain shedding. Here we report a cDNA sequence, encoding a metalloprotease disintegrin, termed ADAM28 (‘a disintegrin and metalloprotease 28’), which was cloned from mouse lung. From protein sequence comparisons, ADAM28 is more closely related to snake venom metalloproteases (SVMPs) than to other ADAMs, and hence may cleave similar substrates to SVMPs, perhaps including components of the extracellular matrix. Northern blot analysis of selected mouse tissues revealed that ADAM28 is expressed highly and in alternatively spliced forms in the epididymis, suggesting a possible role in sperm maturation, and at lower levels in lung. The intracellular maturation of ADAM28 expressed in COS-7 cells resembles that of other ADAMs, in that ADAM28 is made as a precursor and processed to a mature form in a late Golgi compartment of the secretory pathway. Most or all of the mature, and thus presumably catalytically active, form of ADAM28 in COS-7 cells is accessible to cell surface trypsinization, suggesting that ADAM28 functions mainly on the cell surface. A mutation converting the catalytic-site glutamate residue into alanine abolishes pro-domain removal, even though this mutant form of ADAM28 can be transported to the cell surface in a manner similar to the wild-type protein. This suggests that pro-domain removal and maturation of ADAM28 may be, at least in part, autocatalytic. This is in contrast with several other ADAMs, for which furin-like proprotein convertases are involved in pro-domain removal, and in which a glutamate-to-alanine mutation in the catalytic site does not alter pro-domain removal.

scholarly journals Lipid phosphate phosphatases form homo- and hetero-oligomers: catalytic competency, subcellular distribution and function

2008 ◽  
Vol 411 (2) ◽  
pp. 371-377 ◽  
Author(s):  
Jaclyn S. Long ◽  
Nigel J. Pyne ◽  
Susan Pyne
Keyword(s):  
Recombinant Expression ◽  
Catalytic Site ◽  
Wild Type ◽  
Catalytically Active ◽  
Exclusion Chromatography ◽  
Sphingosine 1 Phosphate ◽  
Lipid Phosphate ◽  
Extracellular Side ◽  
And Function ◽  
Lipid Phosphate Phosphatases

Lipid phosphate phosphatases (LPP1–LPP3) have been topographically modelled as monomers (molecular mass of 31–36 kDa) composed of six transmembrane domains and with the catalytic site facing the extracellular side of the plasma membrane or the luminal side of intracellular membranes. The catalytic motif has three conserved domains, termed C1, C2 and C3. The C1 domain may be involved in substrate recognition, whereas C2 and C3 domains appear to participate in the catalytic dephosphorylation of the substrate. We have obtained three lines of evidence to demonstrate that LPPs exist as functional oligomers. First, we have used recombinant expression and immunoprecipitation analysis to demonstrate that LPP1, LPP2 and LPP3 form both homo- and hetero-oligomers. Secondly, large LPP oligomeric complexes that are catalytically active were isolated using gel-exclusion chromatography. Thirdly, we demonstrate that catalytically deficient guinea-pig FLAG-tagged H223L LPP1 mutant can form an oligomer with wild-type LPP1 and that wild-type LPP1 activity is preserved in the oligomer. These findings suggest that, in an oligomeric arrangement, the catalytic site of the wild-type LPP can function independently of the catalytic site of the mutant LPP. Finally, we demonstrate that endogenous LPP2 and LPP3 form homo- and hetero-oligomers, which differ in their subcellular localization and which may confer differing spatial regulation of phosphatidic acid and sphingosine 1-phosphate signalling.

scholarly journals A recombinant human ‘mini’-hexokinase is catalytically active and regulated by hexose 6-phosphates

1992 ◽  
Vol 285 (1) ◽  
pp. 193-199 ◽  
Author(s):  
M Magnani ◽  
M Bianchi ◽  
A Casabianca ◽  
V Stocchi ◽  
A Daniele ◽  
...  
Keyword(s):  
Gene Duplication ◽  
Active Form ◽  
Evolutionary Relationship ◽  
Product Inhibition ◽  
Catalytic Site ◽  
Type I ◽  
Regulatory Site ◽  
Allosteric Site ◽  
Reading Frame ◽  
Catalytically Active

Mammalian hexokinase type I is a 100 kDa enzyme that has been considered to be evolved from an ancestral 50 kDa yeast-type hexokinase, insensitive to product inhibition, by gene duplication and fusion. According to this model, and based on many experimental data, the catalytic site is associated with the C-terminal half of the enzyme, although an allosteric site for the binding of glucose 6-phosphate could be present on the N-terminal half of the molecule. We have isolated a cDNA clone of hexokinase from a lambda gt11 human placenta library comprising 2658 bp, containing a single open reading frame of 1893 nucleotides, which encodes a truncate form of hexokinase starting from asparagine-287 to the terminal serine-917. This clone was further digested with restriction enzyme NcoI to obtain almost only the C-terminal half of human hexokinase starting from methionine-455 to the terminal amino acid and was overexpressed in active form in Escherichia coli and purified by ion-exchange h.p.l.c. The overexpressed ‘mini’-hexokinase was found not only to catalyse glucose phosphorylation, but also to be inhibited by glucose 6-phosphate and other mono- and bis-phosphate sugars exactly like the complete mammalian enzyme. These results suggest that the C-terminal half of human hexokinase, in addition to the catalytic site, also contains the regulatory site and that the evolutionary relationship between the hexokinases should be reconsidered by including the appearance of a regulatory site before the gene duplication.

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