Conformational Dynamics at the Active Site of α-Chymotrypsin and Enzymatic Activity

Agmatine is the product of the decarboxylation of L-arginine by the enzyme arginine decarboxylase. This amine has been attributed to neurotransmitter functions, anticonvulsant, anti-neurotoxic, and antidepressant in mammals and is a potential therapeutic agent for diseases such as Alzheimer’s, Parkinson’s, and cancer. Agmatinase enzyme hydrolyze agmatine into urea and putrescine, which belong to one of the pathways producing polyamines, essential for cell proliferation. Agmatinase from Escherichia coli (EcAGM) has been widely studied and kinetically characterized, described as highly specific for agmatine. In this study, we analyze the amino acids involved in the high specificity of EcAGM, performing a series of mutations in two loops critical to the active-site entrance. Two structures in different space groups were solved by X-ray crystallography, one at low resolution (3.2 Å), including a guanidine group; and other at high resolution (1.8 Å) which presents urea and agmatine in the active site. These structures made it possible to understand the interface interactions between subunits that allow the hexameric state and postulate a catalytic mechanism according to the Mn2+ and urea/guanidine binding site. Molecular dynamics simulations evaluated the conformational dynamics of EcAGM and residues participating in non-binding interactions. Simulations showed the high dynamics of loops of the active site entrance and evidenced the relevance of Trp68, located in the adjacent subunit, to stabilize the amino group of agmatine by cation-pi interaction. These results allow to have a structural view of the best-kinetic characterized agmatinase in literature up to now.

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The Active Site of a Prototypical “Rigid” Drug Target is Marked by Extensive Conformational Dynamics

Angewandte Chemie International Edition ◽

10.1002/anie.202009348 ◽

2020 ◽

Vol 59 (51) ◽

pp. 22916-22921

Author(s):

Himanshu Singh ◽

Chandan K. Das ◽

Suresh K. Vasa ◽

Kristof Grohe ◽

Lars V. Schäfer ◽

...

Keyword(s):

Active Site ◽

Drug Target ◽

Conformational Dynamics

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Protein Immobilization in Metal–Organic Frameworks by Covalent Binding

Australian Journal of Chemistry ◽

10.1071/ch14104 ◽

2014 ◽

Vol 67 (11) ◽

pp. 1629 ◽

Cited By ~ 24

Author(s):

Xuan Wang ◽

Trevor A. Makal ◽

Hong-Cai Zhou

Keyword(s):

Enzymatic Activity ◽

Enzyme Immobilization ◽

Working Conditions ◽

Active Site ◽

Covalent Binding ◽

Metal Organic Frameworks ◽

Protein Immobilization ◽

Immobilization Of Enzymes ◽

Metal Organic ◽

Biological Catalysis

Metal–organic frameworks (MOFs), possessing a well defined system of pores, demonstrate extensive potential serving as a platform in biological catalysis. Successful immobilization of enzymes in a MOF system retains the enzymatic activity, renders the active site more accessible to the substrate, and promises recyclability for reuse, and solvent adaptability in a broad range of working conditions. This highlight describes enzyme immobilization on MOFs via covalent binding and its significance.

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Three Conserved Regions in Baculovirus Sulfhydryl Oxidase P33 Are Critical for Enzymatic Activity and Function

Journal of Virology ◽

10.1128/jvi.01158-17 ◽

2017 ◽

Vol 91 (23) ◽

Cited By ~ 6

Author(s):

Wenhua Kuang ◽

Huanyu Zhang ◽

Manli Wang ◽

Ning-Yi Zhou ◽

Fei Deng ◽

...

Keyword(s):

Enzymatic Activity ◽

Active Site ◽

Salt Bridge ◽

Sulfhydryl Oxidase ◽

Dimer Interface ◽

Conserved Regions ◽

Occlusion Bodies ◽

Budded Virus ◽

And Function ◽

Substrate Proteins

ABSTRACT Baculoviruses encode a conserved sulfhydryl oxidase, P33, which is necessary for budded virus (BV) production and multinucleocapsid occlusion-derived virus (ODV) formation. Here, the structural and functional relationship of P33 was revealed by X-ray crystallography, site-directed mutagenesis, and functional analysis. Based on crystallographic characterization and structural analysis, a series of P33 mutants within three conserved regions, i.e., the active site, the dimer interface, and the R127-E183 salt bridge, were constructed. In vitro experiments showed that mutations within the active site and dimer interface severely impaired the sulfhydryl oxidase activity of P33, while the mutations in the salt bridge had a relatively minor influence. Recombinant viruses containing mutated P33 were constructed and assayed in vivo. Except for the active-site mutant AXXA, all other mutants produced infectious BVs, although certain mutants had a decreased BV production. The active-site mutant H114A, the dimer interface mutant H227D, and the salt bridge mutant R127A-E183A were further analyzed by electron microscopy and bioassays. The occlusion bodies (OBs) of mutants H114A and R127A-E183A had a ragged surface and contained mostly ODVs with a single nucleocapsid. The OBs of all three mutants contained lower numbers of ODVs and had a significantly reduced oral infectivity in comparison to control virus. Crystallographic analyses further revealed that all three regions may coordinate with one another to achieve optimal function of P33. Taken together, our data revealed that all the three conserved regions are involved in P33 activity and are crucial for virus morphogenesis and peroral infectivity. IMPORTANCE Sulfhydryl oxidase catalyzes disulfide bond formation of substrate proteins. P33, a baculovirus-encoded sulfhydryl oxidase, is different from other cellular and viral sulfhydryl oxidases, bearing unique features in tertiary and quaternary structure organizations. In this study, we found that three conserved regions, i.e., the active site, dimer interface, and the R127-E183 salt bridge, play important roles in the enzymatic activity and function of P33. Previous observations showed that deletion of p33 results in a total loss of budded virus (BV) production and in morphological changes in occlusion-derived virus (ODV). Our study revealed that certain P33 mutants lead to occlusion bodies (OBs) with a ragged surface, decreased embedded ODVs, and reduced oral infectivity. Interestingly, some P33 mutants with impaired ODV/OB still retained BV productivity, indicating that the impacts on BV and on ODV/OB are two distinctly different functions of P33, which are likely to be performed via different substrate proteins.

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Coupling between conformational dynamics and catalytic function at the active site of the lead-dependent ribozyme

RNA ◽

10.1261/rna.067579.118 ◽

2018 ◽

Vol 24 (11) ◽

pp. 1542-1554 ◽

Cited By ~ 2

Author(s):

Neil A. White ◽

Minako Sumita ◽

Victor E. Marquez ◽

Charles G. Hoogstraten

Keyword(s):

Active Site ◽

Conformational Dynamics ◽

Catalytic Function

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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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A Single Point Mutation Controls the Rate of Interconversion Between the g+ and g− Rotamers of the Histidine 189 χ2 Angle That Activates Bacterial Enzyme I for Catalysis

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.699203 ◽

2021 ◽

Vol 8 ◽

Author(s):

Jeffrey A. Purslow ◽

Jolene N. Thimmesch ◽

Valeria Sivo ◽

Trang T. Nguyen ◽

Balabhadra Khatiwada ◽

...

Keyword(s):

Protein Design ◽

Active Site ◽

Single Point ◽

Conformational Dynamics ◽

Phosphorylation Site ◽

Phosphoryl Group ◽

Single Point Mutation ◽

Phosphotransferase System ◽

Function Relationship ◽

Enzyme I

Enzyme I (EI) of the bacterial phosphotransferase system (PTS) is a master regulator of bacterial metabolism and a promising target for development of a new class of broad-spectrum antibiotics. The catalytic activity of EI is mediated by several intradomain, interdomain, and intersubunit conformational equilibria. Therefore, in addition to its relevance as a drug target, EI is also a good model for investigating the dynamics/function relationship in multidomain, oligomeric proteins. Here, we use solution NMR and protein design to investigate how the conformational dynamics occurring within the N-terminal domain (EIN) affect the activity of EI. We show that the rotameric g+-to-g− transition of the active site residue His189 χ2 angle is decoupled from the state A-to-state B transition that describes a ∼90° rigid-body rearrangement of the EIN subdomains upon transition of the full-length enzyme to its catalytically competent closed form. In addition, we engineered EIN constructs with modulated conformational dynamics by hybridizing EIN from mesophilic and thermophilic species, and used these chimeras to assess the effect of increased or decreased active site flexibility on the enzymatic activity of EI. Our results indicate that the rate of the autophosphorylation reaction catalyzed by EI is independent from the kinetics of the g+-to-g− rotameric transition that exposes the phosphorylation site on EIN to the incoming phosphoryl group. In addition, our work provides an example of how engineering of hybrid mesophilic/thermophilic chimeras can assist investigations of the dynamics/function relationship in proteins, therefore opening new possibilities in biophysics.

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Kinetic probes for inter-domain co-operation in human somatic angiotensin-converting enzyme

Biochemical Journal ◽

10.1042/bj20050702 ◽

2005 ◽

Vol 391 (3) ◽

pp. 641-647 ◽

Cited By ~ 16

Author(s):

Olga E. Skirgello ◽

Peter V. Binevski ◽

Vladimir F. Pozdnev ◽

Olga A. Kost

Keyword(s):

Angiotensin Converting Enzyme ◽

Enzymatic Activity ◽

Active Site ◽

The Other ◽

Converting Enzyme ◽

Human Enzyme ◽

Independent Functions ◽

The Common ◽

The Individual ◽

Hydrolysis Of

s-ACE (the somatic form of angiotensin-converting enzyme) consists of two homologous domains (N- and C-domains), each bearing a catalytic site. Negative co-operativity between the two domains has been demonstrated for cow and pig ACEs. However, for the human enzyme there are conflicting reports in the literature: some suggest possible negative co-operativity between the domains, whereas others indicate independent functions of the domains within s-ACE. We demonstrate here that a 1:1 stoichiometry for the binding of the common ACE inhibitors, captopril and lisinopril, to human s-ACE is enough to abolish enzymatic activity towards FA {N-[3-(2-furyl)acryloyl]}-Phe-GlyGly, Cbz (benzyloxycarbonyl)-Phe-His-Leu or Hip (N-benzoylglycyl)-His-Leu. The kinetic parameters for the hydrolysis of seven tripeptide substrates by human s-ACE appeared to represent average values for parameters obtained for the individual N- and C-domains. Kinetic analysis of the simultaneous hydrolysis of two substrates, Hip-His-Leu (S1) and Cbz-Phe-His-Leu (S2), with a common product (His-Leu) by s-ACE at different values for the ratio of the initial concentrations of these substrates (i.e. σ=[S2]0/[S1]0) demonstrated competition of these substrates for binding to the s-ACE molecule, i.e. binding of a substrate at one active site makes the other site unavailable for either the same or a different substrate. Thus the two domains within human s-ACE exhibit strong negative co-operativity upon binding of common inhibitors and in the hydrolysis reactions of tripeptide substrates.

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