| 1. |
- Adlercreutz, Patrick
(författare)
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Activation of enzymes in organic media at low water activity by polyols and saccharides
- 1993
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Ingår i: BBA - Protein Structure and Molecular Enzymology. - 0167-4838. ; 1163:2, s. 144-148
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Tidskriftsartikel (refereegranskat)abstract
- Horse liver alcohol dehydrogenase and α-chymotrypsin were deposited on a porous support material, Celite. After equilibration at a well-defined water activity, the catalytic activity was measured with diisopropyl ether as reaction medium. The effects of the presence of polyols and simple saccharides in the preparations were investigated. The additives caused a considerable increase in the amount of water bound to the preparation at a fixed water activity. At low water activities the catalytic activity was increased and at high water activity it was decreased by the additives. The presence of additives increased the ratio of alcoholysis-to-hydrolysis activity of chymotrypsin.
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| 2. |
- Berggren, Kristina, et al.
(författare)
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Substitutions of surface amino acid residues of cutinase probed by aqueous two-phase partitioning
- 2000
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Ingår i: BBA - Protein Structure and Molecular Enzymology. - 0167-4838. ; 1481:2, s. 317-327
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Tidskriftsartikel (refereegranskat)abstract
- The surface properties of a protein are often crucial for recognition and interaction with other molecules. Important functional residues can be identified by mutational analysis. There is a need for rapid methods to study protein surfaces and surface changes due to mutations. Partitioning in aqueous two-phase systems has the potential to be used in this respect since protein partitioning depends on the surface properties of the protein. The influence of surface-exposed amino acid residues in protein partitioning has been studied with cutinase variants, which differed in one or several amino acid residues as a result of site-directed mutagenesis. The solvent accessibility of the mutated residues was determined with a computer program, Graphical Representation and Analysis of Surface Properties. The aqueous two-phase system was composed of dextran and a random copolymer of ethylene oxide and propylene oxide. It was shown, for the first time, to what extent surface-exposed amino acid residues influence the partition coefficient in an aqueous two-phase system. The effect on partitioning could be described only taking into account solvent accessibility and type of residue substitution. The results demonstrate that the system can be used to detect conformational changes in mutant proteins since the expected effect on partitioning due to a mutation can be calculated. The aqueous two-phase system used here does indeed provide a rapid and convenient method to study protein surfaces and slight surface changes due to mutations.
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| 3. |
- Berggren, K, et al.
(författare)
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The surface exposed amino acid residues of monomeric proteins determine the partitioning in aqueous two-phase systems
- 2002
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Ingår i: BBA - Protein Structure and Molecular Enzymology. - 0167-4838. ; 1596:2, s. 253-268
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Tidskriftsartikel (refereegranskat)abstract
- It is of great interest and importance how different amino acid residues contribute to and affect the properties of a protein surface, Partitioning in aqueous two-phase systems has the potential to be used as a rapid and simple method for studying the surface properties of proteins. The influence on partitioning of the surface exposed amino acid residues of eight structurally determined monomeric proteins has been studied. The proteins were characterized in terms of surface exposed residues with a computer program, Graphical Representation and Analysis of Surface Properties (GRASP), and partitioned in two EO30PO70-dextran aqueous two-phase systems, only differing in polymer concentrations (system I: 6.8% EO30PO70, 7.1% dextran; system II: 9% EO30PO70, 9% dextran). We show for the first time that the partitioning behaviour of different monomeric proteins can be described by the differences in surface exposed amino acid residues. The contribution to the partition coefficient of the residues was found to be best characterized by peptide partitioning in the aqueous two-phase system. Compared to hydrophobicity scales available in the literature, each amino acid contribution is characterized by the slope given by the graph of log K against peptide chain length, for peptides of different length containing only one kind of residue. It was also shown that each amino acid contribution is relative to the total protein surface and the other residues on the surface. Surface hydrophobicity calculations realized for systems I and 11 gave respectively correlation coefficients of 0.961 and 0.949 for the linear relation between log K and calculated hydrophobicity values. To study the effect on the partition coefficient of different amino acids, they were grouped into classes according to common characteristics: the presence of an aromatic group, a long aliphatic chain or the presence of charge. Using these groups it was possible to confirm that aromatic residues have the strongest effect on the partition coefficient, giving preference to the upper EO30PO70 phase of the system; on the other hand the presence of charged amino acids on the protein surface enhances the partition of the protein to the lower dextran phase. It is also important to note that the sensitivity of the EO30PO70-dextran system for the surface exposed residues was increased by increasing the polymer concentrations. The partition coefficient of a monomeric protein can thus be predicted from its surface exposed amino acid residues and the system can also be used to characterize protein surfaces of monomeric proteins in general. (C) 2002 Elsevier Science B.V. All rights reserved.
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| 4. |
- Calvet, Silvia, et al.
(författare)
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Enzymatic synthesis of X-Phe-Leu-NH2 in low water content systems : Influence of the N-α protecting group and the reaction medium composition
- 1993
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Ingår i: BBA - Protein Structure and Molecular Enzymology. - 0167-4838. ; 1164:2, s. 189-196
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Tidskriftsartikel (refereegranskat)abstract
- The influence of eight different N-terminal protecting groups (For, Ac, Boc, Fmoc, Mal, Pheac, Aloc and Z) on the α-chymotrypsin-catalyzed synthesis of the dipeptide derivative X-Phe-Leu-NH2 in organic media was studied. Groups such as Ac, For, Boc, Z, Mal, Pheac and Alloc always rendered good peptide yields (92% to 99%) either in acetonitrile or in ethyl acetate. Good correlations were found between molecular and physico-chemical characteristics of the N-α moiety such as the hydrophobicity (log P), ovality and dipole moment and the global reaction rate parameter k′. High k′ values were obtained with the less hydrophobic groups, Ac, For and Mal, that have ovality values close to one and the highest dipole moments. Furthermore, it was found that the relative rate of hydrolysis and aminolysis of the acyl-enzyme intermediate expressed as the partition parameter p is affected by the N-α moiety of the acyl donor. Correlations between this parameter and the dipole moment of the protecting group were observed.
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| 5. |
- Clapés, Pere, et al.
(författare)
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Substrate specificity of α-chymotrypsin-catalyzed esterification in organic media
- 1991
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Ingår i: BBA - Protein Structure and Molecular Enzymology. - : Elsevier BV. - 0167-4838. ; 1118:1, s. 70-76
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Tidskriftsartikel (refereegranskat)abstract
- 11 amino acid derivatives were tested as α-chymotrypsin substrates in the esterification reaction with methanol in organic media. The reactions were carried out in water-saturated ethyl acetate and in acetonitrile containing 4% water. α-Chymotrypsin adsorbed on Celite was used as a catalyst. From initial reaction rate measurements, the Michaelis-Menten parameters Vmax and KM were determine. All the amino acid derivatives tested were esterified and the highest values of kcal KM were obtained with the N-acylated aromatic amino acids. Correlations between Michaelis-Menten parameters and physical properties of the substrates such as molar refractivity (MR) and log P were deduced. The results show that the specificity of the α-chymotrypsin towards the side chain of the amino acids in organic media is the same as that in aqueous media. However, the specificity towards the N-protecting group is opposite to that in water, so the reaction medium affects the interactions of this part of the molecule with the enzyme to a large extent.
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| 6. |
- Egger, Dietlind, et al.
(författare)
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Characterization and optimization of phospholipase A2 catalyzed synthesis of phosphatidylcholine
- 1997
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Ingår i: BBA - Protein Structure and Molecular Enzymology. - 0167-4838. ; 1343:1, s. 76-84
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Tidskriftsartikel (refereegranskat)abstract
- The phospholipase A2 (PLA2) catalyzed synthesis and hydrolysis of phosphatidylcholine (PC) was studied in a water activity controlled organic medium. The aim of the study was to find the conditions most favorable for the synthetic reaction. To do this, the impact of various parameters such as water activity, substrate concentration and temperature on enzyme activity and equilibrium yield was determined. The PC to lysophosphatidylcholine (LPC) ratio at equilibrium increases with decreasing water activity and increasing fatty acid concentration, as can be expected from the law of mass action of an esterification reaction. The enzyme activity on the other hand decreases under conditions that favor the esterification. The best yield in the synthetic reaction is 60% at a water activity of 0.11 and an oleic acid concentration of 1.8 M. That is to our knowledge the highest yield ever reported in this reaction. Both the hydrolysis and synthesis reaction follow Michaelis-Menten kinetics, the apparent K(m) values are the same for PC and LPC, namely 4.9 mM. V(max) is 82.5 and 10.4 nmol h-1 mg-1 for the hydrolysis and synthesis reaction, respectively. Studies on PLA2 at water activity controlled conditions resulted in a more complete understanding of the enzymatic reaction and allowed to find the conditions most favorable for the synthetic reaction.
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| 7. |
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| 8. |
- Gololobov, Mikhail Yu, et al.
(författare)
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Nucleophile specificity in α-chymotrypsin- and subtilisin-(Bacillus subtilis strain 72) catalyzed reactions
- 1992
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Ingår i: BBA - Protein Structure and Molecular Enzymology. - 0167-4838. ; 1160:2, s. 188-192
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Tidskriftsartikel (refereegranskat)abstract
- Nucleophilic properties of amino-acids were studied systematically in acyl-transfer reactions catalyzed by α-chymotrypsin and subtilisin from Bacillus subtilis strain 72 (subtilisin 72) using Mal-l-Ala-l-Ala-l-PheOMe as the acyl-group donor. In α-chymotrypsin-catalyzed reactions, the nucleophile reactivities increase in the following order: d-AlaNH2 < GlyNH2 < l-AlaNH2 < l-ScrNH2 < l-ThrNH2 < l-HisNH2 < l-ValNH2 < l-LcuNH2 < l-TrpNH2 < l-MetNH2 < l-NvaNH2 < l-PheNH2 < l-IleNH2 < l-TyrNH2 < l-ArgNH2. In reactions catalyzed by subtilisin 72, the reactivities increase as follows: l-LeuNH2 < l- IleNH2 < l-ThrNH2 < l-ArgNH2 < l-TrpNH2 < l-NvaNH2 < l-ValNH2 < l-MctNH2 < l-AlaNH2 < l-ScrNH2 < d-AlaNH2 < GlyNH2. In α-chymotrypsin-catalyzed reactions, hydrophobic interactions are entirely responsible for the differences between the reactivity of the nucleophiles for amides of all the amino-acids tested with the exception of d-AlaNH2, l-ArgNH2 and l-TyrNH2. In the reactions catalyzed by subtilisin 72, Amino-acid side-chain characteristics and the nucleophile reactivities are not related. The data obtained show the low selectivity of the S1′ subsite of subtilisin 72 and high specify of this subsite in α-chymotrypsin.
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| 9. |
- Hansson, Mats, et al.
(författare)
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Isolated Bacillus subtilis HemY has coproporphyrinogen III to coproporphyrin III oxidase activity
- 1997
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Ingår i: BBA - Protein Structure and Molecular Enzymology. - 0167-4838. ; 1340:1, s. 97-104
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Tidskriftsartikel (refereegranskat)abstract
- Oxidation of coproporphyrinogen III to coproporphyrin III is found in extracts of Escherichia coli cells containing the Bacillus subtilis HemY protein (M. Hansson and L. Hederstedt, J. Bacteriol. 176, 5962-5970). We have analysed whether this activity is due to the heterologous expression system, since it in vivo would lead to disruption of the heme biosynthetic pathway. B. subtilis hemY was fused in its 3'-end to a polynucleotide encoding six histidine residues and expressed from plasmids in both E. coli and B. subtilis. The His6-tagged HemY protein extracted from membranes using non-ionic detergent was purified by Ni2+ affinity chromatography. Isolated HemY fusion protein synthesised in E. coli and B. subtilis oxidised coproporphyrinogen III to coproporphyrin III. No direct formation of protoporphyrin IX from coproporphyrinogen III could be detected. Our results suggest that the coproporphyrinogen III to coproporphyrin III activity of HemY is either avoided in B. subtilis in vivo or that coproporphyrin III is a heme biosynthetic intermediate in this bacterium.
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| 10. |
- Hansson, Ulla-Britt, et al.
(författare)
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Conformational isomerism of IgG antibodies
- 1997
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Ingår i: BBA - Protein Structure and Molecular Enzymology. - 0167-4838. ; 1340:1, s. 53-62
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Tidskriftsartikel (refereegranskat)abstract
- The purpose of this study was to determine why apparently homogeneous IgG antibodies were, in some cases, fractionated into at least two components by liquid-liquid partition chromatography (LLPC) in an aqueous two-phase system. Four mouse monoclonal IgG antibodies, two against albumin, one against IgG and one against thyroxine, were shown to adopt different conformational isomeric forms. The four antibodies existed in an equilibrium between two or three conformational forms, the proportion of which could also be estimated by LLPC. Since LLPC detects mainly conformational differences within the antigen-binding sites of IgG antibodies, it could be concluded that the conformational forms differed with respect to their combining sites. Moreover, the isomeric forms of an antibody directed against a protein antigen, formed antigen-antibody complexes with almost identical surface properties. In contrast, complexes with different surface properties were formed when the hapten or hapten conjugated to BSA was bound. Thus, both the conformational isomers could bind antigen, at least when the antigen was a small hapten or a hapten conjugated to a carrier protein. Our results suggest that six out of 57 monoclonal IgG antibodies exist in equilibrium between at least two conformational forms and the biological significance of this isomerism is discussed.
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