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On electrostatic effects, minimal motion and other catalytic strategies used by enzymes
KTH, School of Biotechnology (BIO), Biochemistry.ORCID iD: 0000-0002-4066-2776
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Enzymes are powerful biocatalysts that provide rate accelerations of up to 1019 fold compared to the corresponding uncatalyzed reaction in solution. The origin of the remarkable performance displayed by enzymes has fascinated and puzzled researchers for over a hundred years. It is clear that the catalytic effect is a consequence of the higher degree of transition state stabilization for the enzyme catalyzed reaction compared to the corresponding uncatalyzed reaction. It is still not well understood exactly how this transition state stabilization occurs and the relative importance of various catalytic effects are discussed. Catalytic effects involving electrostatics, near attack conformers, dynamic effects and an economy in atomic motion are discussed in this thesis.

The importance of electrostatic effects is corroborated in this thesis. A single hydrogen bond in transition state constitutes an important difference between amidases and esterases. A hydrogen bond in transition state is found in all sixteen analyzed amidases representing ten different reaction mechanisms and eleven different folding families. The hydrogen bond is shown to be either substrate assisted or enzyme assisted. The role of this hydrogen bond is to assist nitrogen inversion in amidases. Esterases lack this interaction in transition state and therefore they are very poor catalysts in the hydrolysis of amides. Electrostatic interactions are found to facilitate proton transfer that enhances the rate of lipase catalyzed N-acylation of amino alcohols.

In this thesis electrostatic effects in the substrate are shown to be important for the lipase catalyzed transacylation of acrylates The α,β-double bond present in acrylates introduce electronic effects that has the consequence of restricting the conformational freedom of the substrate in its ground state to two flat conformations, s-cis and s-trans. It is shown that acrylates form near attack conformers (NACs) from their ground state s-cis/s-trans planar conformations. The ability of the enzyme to accommodate such apparent s-cis/s-trans substrate conformations dictates the probability to form productive transition states and thus the reaction rate.

Dynamic effects are important in enzymes. In this thesis it is found that a point mutation increases the flexibility of a neighbouring residue in Candida antarctica lipase B. This allows the mutated enzyme to explore conformations not accessible for the wild-type enzyme. The dynamics has the effect to decrease steric interactions in transition state with concomitant rate increase for the transacylation of methyl methacrylate.

In this thesis an economy of atomic motion during enzyme catalysis is observed. Nitrogen inversion in amidases constitutes an interesting example. A rotation as part of the reaction mechanism for amide bond hydrolysis would involve much more motion.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology , 2011. , p. 42
Series
Trita-BIO-Report, ISSN 1654-2312 ; 14
National Category
Biochemistry and Molecular Biology
Research subject
SRA - Molecular Bioscience
Identifiers
URN: urn:nbn:se:kth:diva-33334ISBN: 978-91-7415-976-9 (print)OAI: oai:DiVA.org:kth-33334DiVA, id: diva2:414502
Public defence
2011-05-27, D2, Lindstedtsvägen 5, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
QC 20110512Available from: 2011-05-12 Created: 2011-05-03 Last updated: 2022-06-24Bibliographically approved
List of papers
1. Increased activity of enzymatic transacylation of acrylates through rational design of lipases
Open this publication in new window or tab >>Increased activity of enzymatic transacylation of acrylates through rational design of lipases
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2010 (English)In: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 65, no 1-4, p. 3-10Article in journal (Refereed) Published
Abstract [en]

A rational design approach was used to create the mutant Candida antarctica lipase B (CALB, also known as Pseudozyma antarctica lipase B) V190A having a k(cat) three times higher compared to that of the wild type (wt) enzyme for the transacylation of the industrially important compound methyl methacrylate. The enzymatic contribution to the transacylation of various acrylates and corresponding saturated esters was evaluated by comparing the reaction catalysed by CALB wt with the acid (H2SO4) catalysed reaction. The performances of CALB wt and mutants were compared to two other hydrolases, Humicola insolens cutinase and Rhizomucor mihei lipase. The low reaction rates of enzyme catalysed transacylation of acrylates were found to be caused mainly by electronic effects due to the double bond present in this class of molecules. The reduction in rate of enzyme catalysed transacylation of acrylates compared to that of the saturated ester methyl propionate was however less than what could be predicted from the energetic cost of breaking the pi-system of acrylates solely. The nature and concentration of the acyl acceptor was found to have a profound effect on the reaction rate. (C) 2009 Elsevier B.V. All rights reserved.

Keywords
Lipase, CALB, Point mutations, Kinetics, Proficiency
National Category
Biochemistry and Molecular Biology Biochemistry and Molecular Biology Physical Chemistry
Identifiers
urn:nbn:se:kth:diva-29688 (URN)10.1016/j.molcatb.2009.11.016 (DOI)000278926300002 ()2-s2.0-77952673518 (Scopus ID)
Note
QC 20110218Available from: 2011-02-18 Created: 2011-02-11 Last updated: 2024-03-18Bibliographically approved
2. Mutations in the stereospecificity pocket and at the entrance of the active site of Candida antarctica lipase B enhancing enzyme enantioselectivity
Open this publication in new window or tab >>Mutations in the stereospecificity pocket and at the entrance of the active site of Candida antarctica lipase B enhancing enzyme enantioselectivity
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2010 (English)In: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 65, no 1-4, p. 11-17Article in journal (Refereed) Published
Abstract [en]

Two different parts of Candida antarctica lipase B (stereospecificity pocket at the bottom of the active site and hydrophobic tunnel leading to the active site) were redesigned by single- or double-point mutations, in order to better control and improve enzyme enantioselectivity toward secondary alcohols. Single-point isosteric mutations of Ser47 and Thr42 situated in the stereospecificity pocket gave rise to variants with doubled enantioselectivity toward pentan-2-ol, in solid/gas reactor. Besides, the width and shape of the hydrophobic tunnel leading to the active site was modified by producing the following single-point mutants: Ile189Ala, Leu278Val and Ala282Leu. For each of these variants a significant modification of enantioselectivity was observed compared to wild-type enzyme, indicating that discrimination of the enantiomers by the enzyme could also arise from their different accessibilities from the enzyme surface to the catalytic site. (C) 2010 Elsevier B.V. All rights reserved.

Keywords
Lipase B from Candida antarctica, Stereoselective catalysis, Protein engineering, Stereospecificity pocket, Substrate accessibility to the active site
National Category
Biochemistry and Molecular Biology Biochemistry and Molecular Biology Physical Chemistry
Identifiers
urn:nbn:se:kth:diva-29689 (URN)10.1016/j.molcatb.2010.01.007 (DOI)000278926300003 ()2-s2.0-77952583523 (Scopus ID)
Note
QC 20110216Available from: 2011-02-16 Created: 2011-02-11 Last updated: 2024-03-18Bibliographically approved
3. Substrate Conformations Set the Rate of Enzymatic Acrylation by Lipases
Open this publication in new window or tab >>Substrate Conformations Set the Rate of Enzymatic Acrylation by Lipases
2010 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 11, no 6, p. 802-810Article in journal (Refereed) Published
Abstract [en]

Acrylates represent a class of 4-unsaturated compounds of high industrial importance. We investigated the influence of substrate conformations on the experimentally determined reaction rates of the enzyme-catalysed transacylation of methyl acrylate and derivatives by ab initio DFT B3LYP calculations and molecular dynamics simulations. The results supported a least-motion mechanism upon the sp(2) to sp(3) substrate transition to reach the transition state in the enzyme active site. This was in accordance with our hypothesis that acrylates form productive transition states from their low-energy s-sis/s-trans conformations. Apparent k(cat) values were measured for Candida antarctica lipase B (CALB), Humicola insolens cutinase and Rhizomucor miehei lipase and were compared to results from computer simulations. More potent enzymes for acryltransfer, such as the CALB mutant V190A and acrylates with higher turnover numbers, showed elevated populations of productive transition states.

Keywords
acrylate, biocatalysis, enzymes, molecular modeling, protein design
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-27913 (URN)10.1002/cbic.200900758 (DOI)000277299800013 ()20301160 (PubMedID)2-s2.0-77950664710 (Scopus ID)
Note
QC 20110104Available from: 2011-01-04 Created: 2011-01-03 Last updated: 2024-03-18Bibliographically approved
4. Amidases have a hydrogen bond that facilitates nitrogen inversion but esterases have not
Open this publication in new window or tab >>Amidases have a hydrogen bond that facilitates nitrogen inversion but esterases have not
2011 (English)In: ChemCatChem, ISSN 1867-3899, Vol. 3, no 5, p. 853-860Article in journal (Refereed) Published
Abstract [en]

The fact that proteases/amidases can hydrolyze amides efficiently whereas esterases can not has been discussed during the last decades. By using molecular modeling we have found a hydrogen bond in the transition state for protease/amidase catalyzed hydrolysis of peptides and amides donated by the scissile NH-group of the substrate. The hydrogen-bond acceptor was found either in the enzyme (enzyme assisted) or in the substrate (substrate assisted). This new interaction with the NH-hydrogen in the transition state (TS) was found in sixteen proteases/amidases, which represent ten different reaction mechanisms and eleven different folding families. Esterases lack this interaction and, therefore, they are slow in hydrolyzing amides. By mimicking the substrate-assisted catalysis found in amidases we were able to shift reaction specificity of amide over ester synthesis of Candida antarctica lipase B one hundred fold. We propose that the hydrogen bond facilitates nitrogen inversion in amidases.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-33330 (URN)10.1002/cctc.201000448 (DOI)000290445100008 ()2-s2.0-80051810428 (Scopus ID)
Note
QC 20110608Available from: 2011-05-03 Created: 2011-05-03 Last updated: 2024-03-18Bibliographically approved
5. Proton transfer in amino alcohols in transition state of lipase catalyzed N-acylation.
Open this publication in new window or tab >>Proton transfer in amino alcohols in transition state of lipase catalyzed N-acylation.
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(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:kth:diva-33332 (URN)
Note
QS 2011Available from: 2011-05-03 Created: 2011-05-03 Last updated: 2022-06-24Bibliographically approved

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Syrén, Per-Olof

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