Boris Galunsky

Stability of immobilized soybean lipoxygenases: influence of coupling conditions on the ionization state of the active site Fe

The potential application of lipoxygenase as a versatile biocatalyst in enzyme technology is limited by its poor stability. Two types of soybean lipoxygenases, lipoxygenase-1 and -2 (LOX-1 and LOX-2) were purified by a two step anion exchange chromatography. Four different commercially available supports: CNBr Sepharose 4B, Fractogel((R)) EMD Azlactone, Fractogel((R)) EMD Epoxy, and Eupergit((R)) C were tested for immobilization and stabilization of the purified isoenzymes. Both isoenzymes gave good yields in enzyme activity and good stability after immobilization on CNBr Sepharose 4B and Fractogel((R)) EMD Azlactone. Rapid decay in activity associated with change in the ionization state of Fe, as shown by EPR measurements was observed within the first 5 days after immobilization on epoxy activated supports (Eupergit((R)) C and Fractogel((R)) EMD Epoxy) in high ionic strength buffers. Stabilization of the biocatalyst on these supports was achieved by careful adjustment of the immobilization conditions. When immobilized in phosphate buffer of pH 7.5 and low ionic strength (0.05 M), the half-life time of the immobilized enzyme increased 20 fold. The dependence of the stability of LOX immobilized on epoxy activated supports on the coupling conditions was attributed to a modulation of the ligand environment of the iron in the active site and consequently its reactivity.

Proteolytic processing of penicillin amidase from Alcaligenes faecalis cloned in E. coli yields several active forms

Of four enzymatically active forms of Alcaligenes faecalis penicillin amidase (EC 3.5.1.11) observed in sonicated cells, two (PA5.5 and PA5.3; subscript denotes pI) could be isolated and purified in two steps from the cells destroyed by osmotic shock. Active enzyme was only found in the periplasm. PA5.5 converts further to PA5.3 which differs in the molecular mass of the A-chain. The origin of these differences is a conversion of the N-terminal Gln to pyrolideno-carboxilic acid and a loss of three amino acids from the C-terminus of the A-chain. PA5.3 had higher specific activity (10–30%) for the hydrolysis of benzylpenicillin and 6-nitro-3-phenylacetamidobenzoic acid than PA5.5.

Temperature effects on S1- and S1′-enantioselectivity of α-chymotrypsin

Abstract The temperature dependence of E (enantiomeric ratio or enantioselectivity, a quantitative measure for enzyme stereospecificity) has been studied for the α -chymotrypsin catalysed hydrolysis of the enantiomeric N -Boc- l / d -TyrOMe, l / d -TyrOMe, Ac- l / d -PhgOMe, l / d -PhgOMe and for the kinetically controlled synthesis of the diastereomeric dipeptides N -Ac- l -Tyr- l / d -ArgNH 2 and N -Ac- l -Tyr- l / d -ValNH 2 . The results show that the S 1 - and S 1 ′-enantioselectivity can be modulated by the temperature (3–15 fold for the studied substrates in the range 5–45°C). For l / d -PhgOMe a reversal in stereospecificity was found in this temperature interval. For the studied substrates both an increase or decrease of the enantiomeric ratio with increasing temperature was observed. For these processes the following relation for the temperature dependence of E has been derived ln E= ln (k l /k d )=−( ΔΔ H # − ΔΔ H b )/RT+( ΔΔ S # − ΔΔ S b )/R where k l and k d are apparent second order rate constants for the reactions with the l - and d -enantiomers, respectively. ΔΔ denotes the differences between the thermodynamic parameters for transformation of the enantiomeric substrates. The subscript b applies for the binding of the substrate or the nucleophile and the superscript # for the formation of the transition state of the enzyme acylation or deacylation. For the studied processes either the enthalpy (ΔΔ H # −ΔΔ H b ) or the entropy (ΔΔ S # −ΔΔ S b ) term was found to control the discrimination. Thus, the enantioselectivity decreases or increases with temperature, respectively. The influence of ground-state interactions and transition-state stabilisation on enzyme enantioselectivity has been discussed.

GENETIC CONSTRUCTION OF CATALYTICALLY ACTIVE CROSS- SPECIES HETERODIMER PENICILLIN G AMIDASE

SummaryA cross-species penicillin G amidase (PGA) gene (pac) coding for an α-peptide and a linker peptide fromK. citrophila ATCC 21285 and a β-peptide fromE. coli ATCC 11105 has been constructed and cloned inE. coli. The naturally occurring PGA specific processing pathway led to the formation of a hybrid enzyme which was catalytically active. In comparison with the two wild-type enzymes the hybrid PGA was found to have higherkcat values for the three tested substrates benzylpenicillin, ampicillin and 6-nitro-3-phenylacetamido-benzoic acid (NIPAB).Km was between the values of the wild-type enzymes or close to that ofK. citrophila. The presented method for protein design of processed enzymes, like PGA, can be applied to combine enzyme properties from different species for special applications.

Phenylacetyl group as enzyme-cleavable aminoprotection of purine nucleosides

Abstract N 6 -Phenylacetyl-2′-deoxyadenosine and N 2 -Phenylacetyl-2′-deoxyguanosine are readily deprotected in reactions catalyzed by free and immobilized penicillin amidase at pH 7.8 and 25°C.

Do N-terminal nucleophile hydrolases indeed have a single amino acid catalytic center?

A new set of experimental kinetic data on the hydrolysis of a series of phenylacetyl p‐substituted anilides catalyzed by penicillin G acylase from Escherichia coli (PGA) is presented in this article. The Hammett plot of log(kcat,R/kcat,H) versus σp− has three linear segments, which distinguishes the enzyme from the other N‐terminal nucleophile hydrolases for which data are available. Three amino acids in the vicinity of the catalytic SerB1 (AsnB241, AlaB69, and GlnB23) were included in the quantum mechanical model. The stable structures and the transition states for acylation were optimized by molecular mechanical modeling and at the AM1 level of theory for three model substrates (with H, a methoxy group or a nitro group in the para position in the leaving group). Intrinsic interactions of several functional groups at the active site of PGA are discussed in relation to the catalytic efficiency of the enzyme. The energy barrier computed for the first step of acylation (the nucleophilic attack of SerB1) is lower than that for the second step (the collapse of the tetrahedral intermediate). However, the electronic properties of the substituent on the leaving group affect the structure of the second transition state. It is shown that the main chain carbonyl group of GlnB23 forms a hydrogen bond with the leaving group nitrogen, thus influencing the hydrolysis rate. On the basis of our computations, we propose an interpretation of the complex character of the Hammett plot for the reaction catalyzed by PGA. We suggest a modified scheme of the catalytic mechanism in which some of the intramolecular interactions essential for catalysis are included.

The dependency of the stereoselectivity of penicillin amidases-enzymes with R-specific S1- and S-specific S′1-subsites- on temperature and primary structure

SummaryThe stereoselectivity of penicillin amidase (PA, EC 3.5.1.11) from E coli and homologeous enzymes from other sources has been determined as a function of temperature and substrate for hydrolysis and kinetically controlled synthesis. The stereoselectivity of these reactions decreased almost by one order of magnitude from 5 to 45°C. It increased with the substrate (kcat/Km) and nucleophile (kT/kH) specificity, and was found to differ in the S1- (R-specific) and S′1-(S-specific)-binding subsites of the active site. The S1-stereoselectivity was determined mainly by differences in the activation energy, i.e. the turnover number. The stereoselectivity of PA from different sources differed by almost an order of magnitude for the same substrate.

Kinetically VS. Equilibrium-Controlled Synthesis of C—N Bonds in β-Lactams and Peptides with Free and Immobilized Biocatalysts

Hydrolytic enzymes (proteases, penicillin amidase) can be used also for synthetic purposes. The interest to use these enzymes for synthetic purposes as the semisynthesis of β-lactam antibiotics and peptides has increased recently.

Comparative Study of Substrate- and Stereospecificity of Penicillin G Amidases from Different Sources and Hybrid Isoenzymes

Four natural pencillin G amidase variants from different sources and two genetically constructed hybrid enzymes were produced and purified to homogeneity. The specificity constants of one enzyme (E. coli) were found to differ six orders of magnitude for hydrolytic transformations within a wide range of substrates. The substrate specificity of the homologous penicillin amidases was found to differ less than one order of magnitude for hydrolysis of the most specific and up to two orders of magnitude for the less specific substrates. The Si-substrate specificity in hydrolytic and transfer reactions (studied mainly with the E. coli enzyme) varied more than three orders of magnitude for the different substrates. The penicillin amidases were found to be R-specific in the S1-binding site and S-specific in the Si-binding site. The S1-stereoselectivity differs less than one order of magnitude for the different variants. The Si-stereoselectivity is more pronounced, increases with nucleophile specificity, and was found to differ up to three orders of magnitude in transfer reactions for the enzyme from E. coli. The observed variation of enatioselectivity for different penicillin amidases and one substrate can also be achieved by changes in temperature. Comparison of substrate-and stereospecificity of penicillin amidases from different sources and hybrid isoenzymes suggests that similar changes can be expected for enzyme variants derived by rational protein design or directed evolution.

Mass transfer induced interchange of the kinetic and thermodynamic control of enzymic peptide synthesis in biphasic water-organic systems

The α-chymotrypsin catalysed kinetically controlled peptide synthesis in water and in biphasic water-methyl iso-butyl ketone system was compared. Due to the substrate and product partitioning in the biphasic system an interchange of the reaction control was observed at high enzyme concentration. Under these conditions, the rate of mass transfer between the phases was the rate limiting step and the hydrolysis product concentration was found to have a transient maximum ≫ equilibrium value. In this case, most of the peptide was sythetized in a thermodynamically controlled process. In an aqueous one phase system, the peptide synthesis was kinetically controlled.