Cofactor and Cofactor Mimetics

Many natural enzymes need the assistance of protein cofactors to catalyze chemical reactions at a physiologically relevant speed and several of the enzymes that make up for the coagulation cascade are no exception in this regard. Notably, activated factors VII, IX and X display relatively poor enzymatic activity towards their respective macromolecular substrates. The reason for their low proteolytic activity originates from a number of structural restrictions. For instance, not all enzymes are capable to efficiently fold their new amino-terminus into the active site pocket, leaving the catalytic triad immature. Furthermore, serine protease activation is often associated with a reduced plasticity of the protease domain, which improves their proteolytic activity. Nevertheless, some enzymes still require additional stabilization to reduce flexibility of their protease domain. Protein cofactors are designed to optimize the proteolytic activity of such serine proteases, and can improve the catalytic efficiency of these enzymes by one-thousand to one-million fold. The allosteric changes induced by these protein cofactors are specific to each cofactor/enzyme pair. When focusing on the cofactor role of Factor VIIIa (FVIIIa; which stimulates the catalytic activity of factor IXa; FIXa), several aspects are of importance. First, FVIIIa has high affinity for phosphatidylserine-containing phospholipid-membranes, favoring formation of the FVIIIa/FIXa complex at the membrane surface. Being assembled at the membrane surface limits their movements to two dimensions, and enforces the affinity between both proteins. Second, the interactions between FVIIIa and FIXa involve an extended protein surface, which includes interactions between the FVIIIa light chain and FIXa light chain as well as between the FVIIIa A2 domain and the FIXa protease domain. Due to this extended interactive surface, the complex mimics a staked tree, in which FVIIIa orients the FIXa active site at the appropriate distance from the membrane surface. Moreover, binding of the FVIIIa A2 domain to FIXa surface loops reduces flexibility of the protease domain, and it is likely that allosteric changes induced by the A2-domain optimize the conformation of the active site region. Finally, FVIIIa provides also a binding site for the substrate FX. This not only allows FVIIa to function as a molecular bridge between enzyme and substrate, but also helps to align the FX activation peptide with the FIXa active site. This multistep process by which FVIII acts as a cofactor for FIXa may help us to understand how other non-FVIII molecules can be used to stimulate FIXa activity. Several molecular entities have been reported that are enhancing FIXa activity, including short synthetic peptides, monoclonal antibodies and, perhaps best known at this moment, bispecific antibodies that bind both FIXa and FX. Given the complex molecular structure that FVIIIa has and needs to stimulate FIXa activity, it is of interest to reflect on how this translates to the non-FVIII molecules in terms of regulation and potential cofactor activity. Differences in regulation and activity are of particular relevance for laboratory monitoring of these molecules and in the therapeutic setting. Knowing these limitations will help us to optimize the therapeutic application of non-FVIII molecules. Disclosures Lenting: Spark Therapeutics: Honoraria; Catalyst Biosciences: Honoraria; Sobi: Honoraria; Shire/Takeda: Honoraria; NovoNordisk: Honoraria; Biotest: Honoraria; LFB: Honoraria; Roche: Honoraria; laelaps therapeutics: Equity Ownership.

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Mechanism of the CBM35 domain in assisting catalysis by Ape1, a Campylobacter jejuni O-acetyl esterase

10.1101/2021.09.23.461501 ◽

2021 ◽

Author(s):

Chang Sheng-Huei Lin ◽

Ian Y. Yen ◽

Anson C. K. Chan ◽

Michael E. P. Murphy

Keyword(s):

Campylobacter Jejuni ◽

Active Site ◽

Catalytic Efficiency ◽

Binding Mode ◽

Catalytic Triad ◽

Site Directed Mutagenesis ◽

Acetyl Esterase ◽

Lytic Transglycosylases ◽

Active Site Cleft ◽

And Function

AbstractPeptidoglycan (PG) is O-acetylated by bacteria to resist killing by host lysozyme. During PG turnover, however, deacetylation is a prerequisite for glycan strand hydrolysis by lytic transglycosylases. Ape1, a de-O-acetylase from Campylobacter jejuni, is a bi-modular protein composed of an SGNH hydrolase domain and a CBM35 domain. The conserved Asp-His-Ser catalytic triad in the SGNH hydrolase domain confers enzymatic activity. The PG binding mode and function of the CBM35 domain in de-O-acetylation remained unclear. In this paper, we present a 1.8 Å resolution crystal structure of a complex between acetate and Ape1. An active site cleft is formed at the interface of the two domains and two large loops from the CBM35 domain form part of the active site. Site-directed mutagenesis of residues in these loops coupled with activity assays using p-nitrophenol acetate indicate the CBM35 loops are required for full catalytic efficiency. Molecular docking of a model O-acetylated hexasaccharide PG substrate to Ape1 using HADDOCK suggests the interaction is formed by the active cleft and the saccharide motif of PG. Together, we propose that the active cleft of Ape1 diverges from other SGNH hydrolase members by using the CBM35 loops to assist catalysis. The concave Ape1 active cleft may accommodate the long glycan strands for selecting PG substrates to regulate subsequent biological events.

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Contribution of the NH2-terminal EGF-domain of factor IXa to the specificity of intrinsic tenase

Thrombosis and Haemostasis ◽

10.1160/th12-06-0436 ◽

2012 ◽

Vol 108 (12) ◽

pp. 1154-1164 ◽

Cited By ~ 7

Author(s):

Shabir Qureshi ◽

Likui Yang ◽

Alireza Rezaie

Keyword(s):

Active Site ◽

Membrane Surface ◽

Factor Xa ◽

Resonance Energy ◽

Catalytic Efficiency ◽

Normal Activity ◽

Factor X ◽

Factor Ixa ◽

Direct Binding ◽

Egf Domain

SummaryFactor IXa (FIXa) is a vitamin K-dependent coagulation serine protease which binds to factor VIIIa (FVIIIa) on negatively charged phospholipid vesicles (PCPS) to catalyse the activation of factor X (FX) to factor Xa (FXa) in the intrinsic pathway. Fluorescence resonance energy transfer (FRET) studies have indicated that the Gla-domain-dependent interaction of FIXa and FX with PCPS in the presence of FVIIIa positions the active-site of the protease at an appropriate height above the membrane surface to optimise the catalytic reaction. In this study, we investigated the contribution of the NH2-terminal EGF-domain (EGF1) of FIXa to the recognition specificity of intrinsic tenase by constructing an EGF1 deletion mutant of FIXa (FIXa-desEGF1) and characterising the properties of the mutant in kinetic, direct binding and FRET assays. The results of direct binding and kinetic studies demonstrated that the binding affinity of the mutant for interaction with FVIIIa on PCPS has been impaired greater than 10-fold and the catalytic efficiency of the mutant protease FVIIIa-PCPS complex in the activation of FX has been decreased 100-fold. By contrast, the mutant protease exhibited a normal activity toward FX in the absence of the protein cofactor. FRET measurements revealed that the distance of the active-site of the mutant FIXa relative to PCPS vesicles has been decreased 10 Åfrom 75 ±2 Åfor FIXa to 65 ±2 Åfor FIXa-desEGF1 independent of FVIIIa. These results suggest that the NH2-terminal EGF-domain of FIXa provides a binding-site for FVIIIa and plays an essential spacer function in the intrinsic tenase complex.

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Reconstitution of human azurocidin catalytic triad and proteolytic activity by site-directed mutagenesis

Biological Chemistry ◽

10.1515/bc.2008.101 ◽

2008 ◽

Vol 389 (7) ◽

Cited By ~ 2

Author(s):

Mariusz Olczak ◽

Katarzyna Indyk ◽

Teresa Olczak

Keyword(s):

Proteolytic Activity ◽

Active Site ◽

Cleavage Site ◽

Catalytic Triad ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Ph Optimum ◽

Low Efficiency ◽

Primary Cleavage ◽

Secondary Cleavage

AbstractAzurocidin belongs to the serprocidin family, but it is devoid of proteolytic activity due to a substitution of His and Ser residues in the catalytic triad. The aim of this study was to reconstitute the active site of azurocidin by site-directed mutagenesis, analyze its processing and restored proteolytic activity. Azurocidin expressed inSf9 insect cells possessing the reconstituted His41-Asp89-Ser175 triad exhibited significant proteolytic activity toward casein with a pH optimum of approximately 8–9, but a reconstitution of only one active site amino acid did not result in proteolytically active protein. Enzymatically active recombinant azurocidin caused cleavage of the C-terminal fusion tag with the primary cleavage site after lysine at Lys-Leu and after alanine at Ala-Ala, and the secondary cleavage site after arginine at Arg-Gln, as well as with low efficiency caused cleavage of insulin chain B after leucine at Leu-Tyr and Leu-Cys, and after alanine at Ala-Leu. We demonstrate that cleavage of the azurocidin C-terminal tripeptide is not necessary for its enzymatic activity. The first isoleucine present in mature azurocidin can be replaced by similar amino acids, such as leucine or valine, but its substitution by histidine or arginine decreases proteolytic activity.

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Human Prothrombin Variants with Modifications in the Active Site S4 Subpocket Demonstrate Resistance to Dabigatran

Blood ◽

10.1182/blood-2021-150212 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 439-439

Author(s):

Viola J.F. Strijbis ◽

Ka Lei Cheung ◽

Tessa A. Rutten ◽

Pieter H. Reitsma ◽

Daniel Verhoef ◽

...

Keyword(s):

Protein Expression ◽

Serine Protease ◽

Active Site ◽

Serine Proteases ◽

Thrombin Generation ◽

Plasma Concentrations ◽

Catalytic Triad ◽

Wild Type ◽

Protease Domain ◽

Privately Held

Abstract Chymotrypsin-like serine proteases are hallmarked by a protease domain comprising the catalytic triad residues His57, Asp102, and Ser195 (chymotrypsinogen numbering) situated in the active site cleft. While the catalytic triad in conjunction with the oxyanion hole residues regulate substrate cleavage, the active site subpockets (S1-4) control substrate recognition and binding. The high structural homology of the serine protease domains allows for analogous strategies in drug design, which is underscored by the direct oral anticoagulants (DOACs) for the prophylactic management of stroke in atrial fibrillation and prevention and treatment of venous thrombosis. DOACs inhibit coagulation serine proteases by reversibly engaging the active site with high affinity. To expand the repertoire of DOAC-specific reversal agents we have previously successfully modified the S4 active site subpocket of human factor Xa to prevent DOAC binding while preserving catalytic activity [Verhoef 2017 Nature Commun.]. To explore whether an analogous strategy can be applied to create DOAC resistance in the serine protease thrombin, specific substitutions or sequences in or around the dabigatran-binding S4 subsite derived from naturally occurring serine proteases or plasma proteins were introduced in prothrombin. A panel of prothrombin variants was generated and transfected into HEK293 cells to allow for stable protein expression. In some of the generated prothrombin variants comprising insertions in amino acid sequence 91-99 that is directly adjacent to the S4 subsite protein expression was severely impaired. This indicates that exchange of any surface-exposed serine protease or plasma protein region into the prothrombin protease domain is not necessarily compatible with protein expression. In contrast, exchange of the human prothrombin 91-99 sequence for that of human kallikrein 3 or targeted amino acid replacement of S4 subsite residue Ile174 resulted in prothrombin protein expression levels similar to wild-type prothrombin. Following expression, prothrombin variants were purified to homogeneity using the CaptureSelect tm affinity matrix that selects for fully gamma-carboxylated prothrombin. The specific prothrombin clotting activity analyses of the purified prothrombin variants KL3 (0.7 ± 0.2 U/mg), I174A (0.8 ± 0.2 U/mg), and I174F (0.8 ± 0.3 U/mg) demonstrated an overall ~10-fold reduced specific activity relative to wild-type prothrombin (7.5 ± 0.1 U/mg). As such, modification of the S4 subsite likely interferes with the binding and subsequent conversion of fibrinogen by thrombin. To determine whether the prothrombin variants supported tissue factor-initiated thrombin formation in human plasma, prothrombin-deficient plasma was supplemented with increasing plasma concentrations of prothrombin variant (90-180 ug/mL). Consistent with their reduced specific clotting activity, 180 ug/mL prothrombin variant was required to obtain substantial thrombin generation but with reduced thrombin generation parameters (peak thrombin, ETP) relative to supplementation with plasma concentrations of wild-type prothrombin (90 ug/mL). This calibrated automated thrombin generation assay set-up was used to assess the DOAC-resistance of the prothrombin variants. While thrombin formation reached half-maximum inhibition at 532 ± 58 nM dabigatran in wild-type prothrombin-supplemented plasma, addition of the prothrombin variants displayed a ~2-fold reduced sensitivity to dabigatran inhibition (IC50: 1186 ± 136 nM prothrombin-KL3; 851 ± 97 nM prothrombin-I174F; 772 ± 80 nM prothrombin-I174A). This demonstrates that the S4 subsite-modified prothrombin variants are able to support thrombin generation in the presence of physiological plasma concentrations of inhibitor. Collectively, our findings indicate that human prothrombin variants comprising a single point mutation at position Ile174 in the S4 subsite or at a region directly adjacent to the S4 subsite are able to generate thrombin in plasma inhibited by dabigatran. Hence, serine proteases with S4 subpocket modifications have the potential to bypass DOAC therapy and could provide a generic strategy in the development of novel DOAC-bypassing agents. Figure 1 Figure 1. Disclosures Reitsma: VarmX. B.V.: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Patents & Royalties. Verhoef: VarmX. B.V.: Current Employment, Current holder of individual stocks in a privately-held company. Bos: VarmX B.V.: Research Funding; uniQure Biopharma B.V.: Research Funding.

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N-Acetylanthranilate Amidase from Arthrobacter nitroguajacolicus Rü61a, an α/β-Hydrolase-Fold Protein Active towards Aryl-Acylamides and -Esters, and Properties of Its Cysteine-Deficient Variant

Journal of Bacteriology ◽

10.1128/jb.01085-06 ◽

2006 ◽

Vol 188 (24) ◽

pp. 8430-8440 ◽

Cited By ~ 12

Author(s):

Stephan Kolkenbrock ◽

Katja Parschat ◽

Bernd Beermann ◽

Hans-Jürgen Hinz ◽

Susanne Fetzner

Keyword(s):

Conformational Changes ◽

Active Site ◽

Catalytic Properties ◽

Catalytic Efficiency ◽

Catalytic Triad ◽

Kinetic Properties ◽

Conserved Residues ◽

Hydrolase Fold ◽

Secondary Structure Predictions ◽

Amide Hydrolase

ABSTRACT N-acetylanthranilate amidase (Amq), a 32.8-kDa monomeric amide hydrolase, is involved in quinaldine degradation by Arthrobacter nitroguajacolicus Rü61a. Sequence analysis and secondary structure predictions indicated that Amq is related to carboxylesterases and belongs to the α/β-hydrolase-fold superfamily of enzymes; inactivation of (His6-tagged) Amq by phenylmethanesulfonyl fluoride and diethyl pyrocarbonate and replacement of conserved residues suggested a catalytic triad consisting of S155, E235, and H266. Amq is most active towards aryl-acetylamides and aryl-acetylesters. Remarkably, its preference for ring-substituted analogues was different for amides and esters. Among the esters tested, phenylacetate was hydrolyzed with highest catalytic efficiency (k cat/Km = 208 mM−1 s−1), while among the aryl-acetylamides, o-carboxy- or o-nitro-substituted analogues were preferred over p-substituted or unsubstituted compounds. Hydrolysis by His6Amq of primary amides, lactams, N-acetylated amino acids, azocoll, tributyrin, and the acylanilide and urethane pesticides propachlor, propham, carbaryl, and isocarb was not observed; propanil was hydrolyzed with 1% N-acetylanthranilate amidase activity. The catalytic properties of the cysteine-deficient variant His6AmqC22A/C63A markedly differed from those of His6Amq. The replacements effected some changes in Km s of the enzyme and increased k cats for most aryl-acetylesters and some aryl-acetylamides by factors of about three to eight while decreasing k cat for the formyl analogue N-formylanthranilate by several orders of magnitude. Circular dichroism studies indicated that the cysteine-to-alanine replacements resulted in significant change of the overall fold, especially an increase in α-helicity of the cysteine-deficient protein. The conformational changes may also affect the active site and may account for the observed changes in kinetic properties.

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Essential role of the disintegrin-like domain in ADAMTS13 function

Blood ◽

10.1182/blood-2008-11-187914 ◽

2009 ◽

Vol 113 (22) ◽

pp. 5609-5616 ◽

Cited By ~ 64

Author(s):

Rens de Groot ◽

Ajoy Bardhan ◽

Nalisha Ramroop ◽

David A. Lane ◽

James T. B. Crawley

Keyword(s):

Active Site ◽

Catalytic Efficiency ◽

Targeted Mutagenesis ◽

Fold Reduction ◽

Scissile Bond ◽

And Function ◽

Von Willebrand ◽

A2 Domain ◽

Willebrand Factor

ADAMTS13 is a highly specific multidomain plasma metalloprotease that regulates the multimeric size and function of von Willebrand factor (VWF) through cleavage at a single site in the VWF A2 domain. The precise role that the ADAMTS13 disintegrin-like domain plays in its function remains uncertain. Truncated ADAMTS13 variants suggested the importance of the disintegrin-like domain for both enzyme activity and specificity. Targeted mutagenesis of nonconserved regions (among ADAMTS family members) in the disintegrin-like domain identified 3 of 8 ADAMTS13 mutants (R349A, L350G, V352G) with reduced proteolytic activity. Kinetic analyses revealed a 5- to 20-fold reduction in catalytic efficiency of VWF115 (VWF residues 1554-1668) proteolysis by these mutants. These residues form a predicted exposed exosite on the surface of the disintegrin-like domain that lies approximately 26 Å from the active site. Kinetic analysis of VWF115 carrying the D1614A mutation suggested that Arg349 in the ADAMTS13 disintegrin-like domain interacts directly with Asp1614 in VWF A2. We hypothesize that this interaction assists in positioning the scissile bond within the active site of ADAMTS13 and therefore plays a major role in determining cleavage parameters (Km and kcat), as opposed to binding affinity (Kd) of ADAMTS13 for VWF, the latter being primarily determined by the spacer domain.

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An Enzyme-Linked Immunosorbent Assay for Urokinase-Type Plasminogen Activator (u-PA) and Mutants and Chimeras Containing the Serine Protease Domain of u-PA

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648387 ◽

1992 ◽

Vol 67 (01) ◽

pp. 095-100 ◽

Cited By ~ 18

Author(s):

Paul J Declerck ◽

Leen Van Keer ◽

Maria Verstreken ◽

Désiré Collen

Keyword(s):

Serine Protease ◽

Plasminogen Activator ◽

Active Site ◽

Enzyme Linked Immunosorbent Assay ◽

Single Chain ◽

Protease Domain ◽

Serine Protease Domain ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator ◽

Normal Human

SummaryAn enzyme-linked immunosorbent assay (ELISA) for quantitation of natural and recombinant plasminogen activators containing the serine protease domain (B-chain) of urokinase-type plasminogen activator (u-PA) was developed, based on two murine monoclonal antibodies, MA-4D1E8 and MA-2L3, raised against u-PA and reacting with non-overlapping epitopes in the B-chain. MA-4D1E8 was coated on microtiter plates and bound antigen was quantitated with MA-2L3 conjugated with horseradish peroxidase. The intra-assay, inter-assay and inter-dilution coefficients of variation of the assay were 6%, 15% and 9%, respectively. Using recombinant single-chain u-PA (rscu-PA) as a standard, the u-PA-related antigen level in normal human plasma was 1.4 ± 0.6 ng/ml (mean ± SD, n = 27).The ELISA recognized the following compounds with comparable sensitivity: intact scu-PA (amino acids, AA, 1 to 411), scu-PA-32k (AA 144 to 411), a truncated (thrombin-derived) scu-PA comprising A A 157 to 411, and chimeric t-PA/u-PA molecules including t-PA(AA1-263)/scu-PA(AA144-411), t-PA(AA1-274)/scu-PA(AA138-411) and t-PA(AA87-274)/scu-PA(AA138-411). Conversion of single-chain to two-chain forms of u-PA or inhibition of active two-chain forms with plasminogen activator inhibitor-1 or with the active site serine inhibitor phenyl-methyl-sulfonyl fluoride, did not alter the reactivity in the assay. In contrast, inactivation with α2-antiplasmin or with the active site histidine inhibitor Glu-Gly-Arg-CH2Cl resulted in a 3- to 5-fold reduction of the reactivity. When purified scu-PA-32k was added to pooled normal human plasma at final concentrations ranging from 20 to 1,000 ng/ml, recoveries in the ELISA were between 84 and 110%.The assay was successfully applied for the quantitation of pharmacological levels of scu-PA and t-PA(AA87_274)/scu-PA(AA138-411) in plasma during experimental thrombolysis in baboons.Thus the present ELISA, which is specifically dependent on the presence of the serine protease part of u-PA, is useful for measurement of a wide variety of variants and chimeras of u-PA which are presently being developed for improved thrombolytic therapy.

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Kinetic studies and homology modeling of a dual-substrate linalool/nerolidol synthase from Plectranthus amboinicus

Scientific Reports ◽

10.1038/s41598-021-96524-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Nur Suhanawati Ashaari ◽

Mohd Hairul Ab. Rahim ◽

Suriana Sabri ◽

Kok Song Lai ◽

Adelene Ai-Lian Song ◽

...

Keyword(s):

Active Site ◽

Catalytic Efficiency ◽

Kinetic Studies ◽

Homology Model ◽

Biochemical Properties ◽

Terpene Synthase ◽

Monoterpene Synthase ◽

Terminal Domain ◽

Catalytic Active Site ◽

Plectranthus Amboinicus

AbstractLinalool and nerolidol are terpene alcohols that occur naturally in many aromatic plants and are commonly used in food and cosmetic industries as flavors and fragrances. In plants, linalool and nerolidol are biosynthesized as a result of respective linalool synthase and nerolidol synthase, or a single linalool/nerolidol synthase. In our previous work, we have isolated a linalool/nerolidol synthase (designated as PamTps1) from a local herbal plant, Plectranthus amboinicus, and successfully demonstrated the production of linalool and nerolidol in an Escherichia coli system. In this work, the biochemical properties of PamTps1 were analyzed, and its 3D homology model with the docking positions of its substrates, geranyl pyrophosphate (C10) and farnesyl pyrophosphate (C15) in the active site were constructed. PamTps1 exhibited the highest enzymatic activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, and in the presence of 20 mM magnesium as a cofactor. The Michaelis–Menten constant (Km) and catalytic efficiency (kcat/Km) values of 16.72 ± 1.32 µM and 9.57 × 10–3 µM−1 s−1, respectively, showed that PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The PamTps1 exhibits feature of a class I terpene synthase fold that made up of α-helices architecture with N-terminal domain and catalytic C-terminal domain. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity, whilst residues from the RRx8W motif, RxR motif, H-α1 and J-K loops formed the active site lid that shielded the highly reactive carbocationic intermediates from the solvents. The dual substrates use by PamTps1 was hypothesized to be possible due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by the protein modelling and docking analysis. This model serves as a first glimpse into the structural insights of the PamTps1 catalytic active site as a multi-substrate linalool/nerolidol synthase.

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Effects of viscosity and osmotic stress on the reaction of human butyrylcholinesterase with cresyl saligenin phosphate, a toxicant related to aerotoxic syndrome: kinetic and molecular dynamics studies

Biochemical Journal ◽

10.1042/bj20130389 ◽

2013 ◽

Vol 454 (3) ◽

pp. 387-399 ◽

Cited By ~ 34

Author(s):

Patrick Masson ◽

Sofya Lushchekina ◽

Lawrence M. Schopfer ◽

Oksana Lockridge

Keyword(s):

Osmotic Stress ◽

Osmotic Pressure ◽

Active Site ◽

Md Simulations ◽

Catalytic Triad ◽

Wild Type ◽

Irreversible Inhibitor ◽

Enzyme Active Site ◽

Diffusion Controlled ◽

Peripheral Site

CSP (cresyl saligenin phosphate) is an irreversible inhibitor of human BChE (butyrylcholinesterase) that has been involved in the aerotoxic syndrome. Inhibition under pseudo-first-order conditions is biphasic, reflecting a slow equilibrium between two enzyme states E and E′. The elementary constants for CSP inhibition of wild-type BChE and D70G mutant were determined by studying the dependence of inhibition kinetics on viscosity and osmotic pressure. Glycerol and sucrose were used as viscosogens. Phosphorylation by CSP is sensitive to viscosity and is thus strongly diffusion-controlled (kon≈108 M−1·min−1). Bimolecular rate constants (ki) are about equal to kon values, making CSP one of the fastest inhibitors of BChE. Sucrose caused osmotic stress because it is excluded from the active-site gorge. This depleted the active-site gorge of water. Osmotic activation volumes, determined from the dependence of ki on osmotic pressure, showed that water in the gorge of the D70G mutant is more easily depleted than that in wild-type BChE. This demonstrates the importance of the peripheral site residue Asp70 in controlling the active-site gorge hydration. MD simulations provided new evidence for differences in the motion of water within the gorge of wild-type and D70G enzymes. The effect of viscosogens/osmolytes provided information on the slow equilibrium E⇌E′, indicating that alteration in hydration of a key catalytic residue shifts the equilibrium towards E′. MD simulations showed that glycerol molecules that substitute for water molecules in the enzyme active-site gorge induce a conformational change in the catalytic triad residue His438, leading to the less reactive form E′.

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Dynamism and Cooperativity Essential for Efficient Catalysis in Aspergillus Niger Monoamine Oxidase

10.26434/chemrxiv.7707857.v1 ◽

2019 ◽

Author(s):

Christian Curado-Carballada ◽

Ferran Feixas ◽

Sílvia Osuna

Keyword(s):

Aspergillus Niger ◽

Monoamine Oxidase ◽

Active Site ◽

Conformational Dynamics ◽

Catalytic Efficiency ◽

Building Blocks ◽

Chiral Amine ◽

Evolutionary Pathway ◽

Accelerated Molecular Dynamics ◽

Open States

Aspergillus niger Monoamine Oxidase (MAO-N) is a homodimeric enzyme responsible for the oxidation of amines into the corresponding imine. Laboratory evolved variants of MAO-N in combination with a non-selective chemical reductant represents a powerful strategy for the deracemisation of chiral amine mixtures and, thus, is of interest for obtaining chiral amine building blocks. MAO-N presents a rich conformational dynamics with a flexible ß-hairpin region that can adopt closed, partially closed and open states. Despite the ß-hairpin conformational dynamics is altered along the laboratory evolutionary pathway of MAO-N, the connection between the ß-hairpin conformational dynamics and active site catalysis still remains unclear. In this work, we use accelerated molecular dynamics to elucidate the potential interplay between the ß-hairpin conformational dynamics and catalytic activity in MAO-N wild type and its evolved D5 variant. Our study reveals a delicate communication between both MAO-N subunits that impacts the active site architecture, and thus its catalytic efficiency. In both MAO-N WT and the laboratory evolved D5 variant, the ß-hairpin conformation in one of the monomers affects the productive binding of the substrate in the active site of the other subunit. However, both MAO-N WT and D5 variants show a quite different behaviour due to the distal mutations introduced experimentally with Directed Evolution.

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