A rate expression for enzymatic reactions. The dynamical relation between coupled oscillators at the active site and the rate enhancement of enzyme-catalysed processes

2001 ◽  
Vol 3 (18) ◽  
pp. 4072-4081 ◽  
Author(s):  
Carlo Canepa ◽  
Robert D. Bach
Keyword(s):  
Active Site ◽  
Coupled Oscillators ◽  
Enzymatic Reactions ◽  
Rate Enhancement ◽  
Rate Expression ◽  
Dynamical Relation

scholarly journals Chi hotspot control of RecBCD helicase-nuclease by long-range intramolecular signaling

2020 ◽  
Author(s):  
Susan K. Amundsen ◽  
Andrew F. Taylor ◽  
Gerald R. Smith
Keyword(s):  
Active Site ◽  
Recognition Site ◽  
Dna Unwinding ◽  
Helicase Domain ◽  
Enzymatic Reactions ◽  
Deficient Mutant ◽  
Long Distance ◽  
Intimate Contact ◽  
Recombination Hotspots ◽  
Dependent Signal

AbstractRepair of broken DNA by homologous recombination requires coordinated enzymatic reactions to prepare it for interaction with intact DNA. The multiple activities of enterobacterial RecBCD helicase-nuclease are coordinated by Chi recombination hotspots (5’ GCTGGTGG 3’) recognized during DNA unwinding. Chi is recognized in a tunnel in RecC but activates the RecB nuclease, >25 Ǻ away. How the Chi-dependent signal travels this long distance has been unknown. We found a Chi-recognition-deficient mutant in the RecB helicase domain located >45 Ǻ from both the Chi-recognition site and the nuclease active site. This unexpected observation led us to find additional mutations that reduced or eliminated Chi hotspot activity in each subunit and widely scattered throughout RecBCD. Each mutation alters the intimate contact between one or another pair of subunits in the crystal or cryoEM structures of RecBCD bound to DNA. Collectively, these mutations span a path ∼185 Ǻ long from the Chi recognition site to the nuclease active site. We discuss these surprising results in the context of an intramolecular signal transduction accounting for many previous observations.

Probing catalytic rate enhancement during intramembrane proteolysis

2016 ◽  
Vol 397 (9) ◽  
pp. 907-919 ◽  
Author(s):  
Elena Arutyunova ◽  
Cameron C. Smithers ◽  
Valentina Corradi ◽  
Adam C. Espiritu ◽  
Howard S. Young ◽  
...  
Keyword(s):  
Active Site ◽  
Serine Proteases ◽  
Homology Model ◽  
Intramembrane Proteolysis ◽  
Oxyanion Hole ◽  
Rate Enhancement ◽  
Transition State Stabilization ◽  
The Family ◽  
Catalytic Dyad

Abstract Rhomboids are ubiquitous intramembrane serine proteases involved in various signaling pathways. While the high-resolution structures of the Escherichia coli rhomboid GlpG with various inhibitors revealed an active site comprised of a serine-histidine dyad and an extensive oxyanion hole, the molecular details of rhomboid catalysis were unclear because substrates are unknown for most of the family members. Here we used the only known physiological pair of AarA rhomboid with its psTatA substrate to decipher the contribution of catalytically important residues to the reaction rate enhancement. An MD-refined homology model of AarA was used to identify residues important for catalysis. We demonstrated that the AarA active site geometry is strict and intolerant to alterations. We probed the roles of H83 and N87 oxyanion hole residues and determined that substitution of H83 either abolished AarA activity or reduced the transition state stabilization energy (ΔΔG‡) by 3.1 kcal/mol; substitution of N87 decreased ΔΔG‡ by 1.6–3.9 kcal/mol. Substitution M154, a residue conserved in most rhomboids that stabilizes the catalytic general base, to tyrosine, provided insight into the mechanism of nucleophile generation for the catalytic dyad. This study provides a quantitative evaluation of the role of several residues important for hydrolytic efficiency and oxyanion stabilization during intramembrane proteolysis.

scholarly journals Modelling the Effects of Ageing Time of Starch on the Enzymatic Activity of Three Amylolytic Enzymes

2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
Nelson P. Guerra ◽  
Lorenzo Pastrana Castro
Keyword(s):  
Enzymatic Activity ◽  
Conformational Changes ◽  
Active Site ◽  
Ageing Time ◽  
Enzymatic Reactions ◽  
Reaction Products ◽  
Inhibitory Effects ◽  
Amylolytic Enzymes ◽  
Modified Forms

The effect of increasing ageing time (t) of starch on the activity of three amylolytic enzymes (Termamyl, San Super, and BAN) was investigated. Although all the enzymatic reactions follow michaelian kinetics,vmaxdecreased significantly (P<0.05) andKMincreased (although not always significantly) with the increase int. The conformational changes produced in the starch chains as a consequence of the ageing seemed to affect negatively the diffusivity of the starch to the active site of the enzymes and the release of the reaction products to the medium. A similar effect was observed when the enzymatic reactions were carried out with unaged starches supplemented with different concentrations of gelatine [G]. The inhibition in the amylolytic activities was best mathematically described by using three modified forms of the Michaelis-Menten model, which included a term to consider, respectively, the linear, exponential, and hyperbolic inhibitory effects oftand [G].

scholarly journals Rate-enhancing Single Amino Acid Mutation for Hydrolases: A Statistical Profiling

2021 ◽  
Author(s):  
Bailu Yan ◽  
Xinchun Ran ◽  
Yaoyukun Jiang ◽  
Sarah K. Torrence ◽  
Li Yuan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Center Of Mass ◽  
Hydrocarbon Chain ◽  
Data Bank ◽  
Single Amino Acid ◽  
Spatial Proximity ◽  
Rate Enhancement ◽  
Percentile Rank ◽  
Rate Acceleration

We reported the statistical profiling for rate-enhancing mutant hydrolases with single amino acid substitution. We constructed an integrated structure-kinetics database, IntEnzyDB, which contains 3,907 experimentally characterized hydrolase kinetics and 2,715 hydrolase Protein Data Bank IDs. The hydrolase kinetics data involve 9% rate-enhancing mutations. Mutation to nonpolar residues with a hydrocarbon chain shows a stronger preference for rate acceleration than to polar or charged residues. To elucidate the structure-kinetics relationship for rate-enhancing mutations, we categorized each mutation into one of the three spatial shells of hydrolases. We defined the spatial shells by reference to either the active site or the center-of-mass of the enzyme. In either case, mutations in the first shell (i.e., closest to the reference point) appear on average more rate-deleterious than those in the other two shells (i.e., ~1.0 kcal/mol in ∆∆G‡ ). Under the active-site reference, mutations in the third shell (i.e., most distal to the active site) exhibit the highest likelihood of rate enhancement. This propensity is significant for larger-sized hydrolases. In contrast, under the center-of-mass reference, mutations in the second shell (i.e., 33.3th to 66.7th percentile rank of spatial proximity to the center-of-mass of the enzyme) show the highest likelihood of rate enhancement. This trend is significant for smaller-sized hydrolases. The studies reveal the statistical features for identifying rate-enhancing mutations in hydrolases, which will potentially guide hydrolase discovery in biocatalysis.

scholarly journals Rate-enhancing Single Amino Acid Mutation for Hydrolases: A Statistical Profiling

2021 ◽  
Author(s):  
Bailu Yan ◽  
Xinchun Ran ◽  
Yaoyukun Jiang ◽  
Sarah K. Torrence ◽  
Li Yuan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Center Of Mass ◽  
Hydrocarbon Chain ◽  
Data Bank ◽  
Single Amino Acid ◽  
Spatial Proximity ◽  
Rate Enhancement ◽  
Percentile Rank ◽  
Rate Acceleration

We reported the statistical profiling for rate-enhancing mutant hydrolases with single amino acid substitution. We constructed an integrated structure-kinetics database, IntEnzyDB, which contains 3,907 experimentally characterized hydrolase kinetics and 2,715 hydrolase Protein Data Bank IDs. The hydrolase kinetics data involve 9% rate-enhancing mutations. Mutation to nonpolar residues with a hydrocarbon chain shows a stronger preference for rate acceleration than to polar or charged residues. To elucidate the structure-kinetics relationship for rate-enhancing mutations, we categorized each mutation into one of the three spatial shells of hydrolases. We defined the spatial shells by reference to either the active site or the center-of-mass of the enzyme. In either case, mutations in the first shell (i.e., closest to the reference point) appear on average more rate-deleterious than those in the other two shells (i.e., ~1.0 kcal/mol in ∆∆G‡ ). Under the active-site reference, mutations in the third shell (i.e., most distal to the active site) exhibit the highest likelihood of rate enhancement. This propensity is significant for larger-sized hydrolases. In contrast, under the center-of-mass reference, mutations in the second shell (i.e., 33.3th to 66.7th percentile rank of spatial proximity to the center-of-mass of the enzyme) show the highest likelihood of rate enhancement. This trend is significant for smaller-sized hydrolases. The studies reveal the statistical features for identifying rate-enhancing mutations in hydrolases, which will potentially guide hydrolase discovery in biocatalysis.

scholarly journals Chi hotspot control of RecBCD helicase-nuclease by long-range intramolecular signaling

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Susan K. Amundsen ◽  
Andrew F. Taylor ◽  
Gerald R. Smith
Keyword(s):  
Active Site ◽  
Recognition Site ◽  
Dna Unwinding ◽  
Helicase Domain ◽  
Enzymatic Reactions ◽  
Deficient Mutant ◽  
Long Distance ◽  
Intimate Contact ◽  
Recombination Hotspots ◽  
Dependent Signal

Abstract Repair of broken DNA by homologous recombination requires coordinated enzymatic reactions to prepare it for interaction with intact DNA. The multiple activities of enterobacterial RecBCD helicase-nuclease are coordinated by Chi recombination hotspots (5′ GCTGGTGG 3′) recognized during DNA unwinding. Chi is recognized in a tunnel in RecC but activates the RecB nuclease, > 25 Ǻ away. How the Chi-dependent signal travels this long distance has been unknown. We found a Chi hotspot-deficient mutant in the RecB helicase domain located > 45 Ǻ from both the Chi-recognition site and the nuclease active site. This unexpected observation led us to find additional mutations that reduced or eliminated Chi hotspot activity in each subunit and widely scattered throughout RecBCD. Each mutation alters the intimate contact between one or another pair of subunits in crystal or cryoEM structures of RecBCD bound to DNA. Collectively, these mutations span a path about 185 Ǻ long from the Chi recognition site to the nuclease active site. We discuss these surprising results in the context of an intramolecular signal transduction accounting for many previous observations.

Rationalization of enzyme enantioselectivity by active-site cubic-space models

2002 ◽  
Vol 80 (6) ◽  
pp. 559-564 ◽  
Author(s):  
Gianluca Ottolina ◽  
Giacomo Carrea
Keyword(s):  
Active Site ◽  
Enzymatic Reactions

Active-site models of enzymes are important tools that help researchers in understanding and predicting the stereochemical outcome of enzymatic reactions. In this article, various active-site models for dehydrogenases, hydrolases, oxygenases, and oxynitrilases have been reviewed.

scholarly journals Contribution of active site glutamine to rate enhancement in ubiquitin C-terminal hydrolases

FEBS Journal ◽  
2012 ◽  
Vol 279 (6) ◽  
pp. 1106-1118 ◽  
Author(s):  
David A. Boudreaux ◽  
Joseph Chaney ◽  
Tushar K. Maiti ◽  
Chittaranjan Das
Keyword(s):  
Active Site ◽  
Rate Enhancement

scholarly journals Crystal Structure of Bacterial Cystathionine Γ-Lyase in The Cysteine Biosynthesis Pathway of Staphylococcus aureus

Crystals ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 656 ◽  
Author(s):  
Dukwon Lee ◽  
Soyeon Jeong ◽  
Jinsook Ahn ◽  
Nam-Chul Ha ◽  
Ae-Ran Kwon
Keyword(s):  
Crystal Structure ◽  
Staphylococcus Aureus ◽  
Active Site ◽  
Structural Changes ◽  
Enzymatic Reactions ◽  
Biosynthesis Pathway ◽  
Active Site Residues ◽  
Structural Comparison ◽  
General Base ◽  
Bond Network

Many enzymes require pyridoxal 5’-phosphate (PLP) as an essential cofactor and share active site residues in mediating diverse enzymatic reactions. Methionine can be converted into cysteine by cystathionine γ-lyases (CGLs) through a transsulfuration reaction dependent on PLP. In bacteria, MccB, also known as YhrB, exhibits CGL activity that cleaves the C–S bond of cystathionine at the γ position. In this study, we determined the crystal structure of MccB from Staphylococcus aureus in its apo- and PLP-bound forms. The structures of MccB exhibited similar molecular arrangements to those of MetC-mediating β-elimination with the same substrate and further illustrated PLP-induced structural changes in MccB. A structural comparison to MetC revealed a longer distance between the N-1 atom of the pyridine ring of PLP and the Oδ atom of the Asp residue, as well as a wider and more flexible active site environment in MccB. We also found a hydrogen bond network in Ser-water-Ser-Glu near the Schiff base nitrogen atom of the PLP molecule and propose the Ser-water-Ser-Glu motif as a general base for the γ-elimination process. Our study suggests the molecular mechanism for how homologous enzymes that use PLP as a cofactor catalyze different reactions with the same active site residues.

scholarly journals Neutron crystallography of copper amine oxidase reveals keto/enolate interconversion of the quinone cofactor and unusual proton sharing

2020 ◽  
Vol 117 (20) ◽  
pp. 10818-10824 ◽  
Author(s):  
Takeshi Murakawa ◽  
Kazuo Kurihara ◽  
Mitsuo Shoji ◽  
Chie Shibazaki ◽  
Tomoko Sunami ◽  
...  
Keyword(s):  
Active Site ◽  
Amine Oxidase ◽  
Catalytic Reactions ◽  
Enzymatic Reactions ◽  
Histidine Residue ◽  
Protonation State ◽  
Copper Amine Oxidase ◽  
Site Structure ◽  
Active Site Structure ◽  
Topa Quinone

Recent advances in neutron crystallographic studies have provided structural bases for quantum behaviors of protons observed in enzymatic reactions. Thus, we resolved the neutron crystal structure of a bacterial copper (Cu) amine oxidase (CAO), which contains a prosthetic Cu ion and a protein-derived redox cofactor, topa quinone (TPQ). We solved hitherto unknown structures of the active site, including a keto/enolate equilibrium of the cofactor with a nonplanar quinone ring, unusual proton sharing between the cofactor and the catalytic base, and metal-induced deprotonation of a histidine residue that coordinates to the Cu. Our findings show a refined active-site structure that gives detailed information on the protonation state of dissociable groups, such as the quinone cofactor, which are critical for catalytic reactions.

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