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The catalytic consequences of experimental evolution. Studies on the subunit structure of the second (ebg) beta-galactosidase of Escherichia coli, and on catalysis by ebgab, an experimental evolvant containing two amino acid substitutions.

ABSTRACT: 1. The ratio of ebgA-gene product of ebgC-gene product in the functional aggregate of ebg beta-galactosidases was determined to be 1:1 by isolation of the enzyme from bacteria grown on uniformly radiolabelled amino acids and separation of the subunits by gel-permeation chromatography under denaturing conditions. 2. This datum, taken together with a recalculation of the previous ultracentrifuge data [Hall (1976) J. Mol. Biol. 107, 71-84], analytical gel-permeation chromatography and electron microscopy, strongly suggests an alpha 4 beta 4 quaternary structure for the enzyme. 3. The second chemical step in the enzyme turnover sequence, hydrolysis of the galactosyl-enzyme intermediate, is markedly slower for ebgab, having both Asp-97----Asn and Trp-977----Cys changes in the large subunit, than for ebga (having only the first change) and ebgb (having only the second), and is so slow as to be rate-determining even for an S-glycoside, beta-D-galactopyranosyl thiopicrate, as is shown by nucleophilic competition with methanol. 4. The selectivity of galactosyl-ebgab between water and methanol on a molar basis is 57, similar to the value for galactosyl-ebgb. 5. The equilibrium constant for the hydrolysis of lactose at 37 degrees C is 152 +/- 19 M, that for hydrolysis of allolactose is approx. 44 M and that for hydrolysis of lactulose is approx. 40 M. 6. A comparison of the free-energy profiles for the hydrolyses of lactose catalysed by the double mutant with those for the wild-type and the single mutants reveals that free-energy changes from the two mutations are not in general independently additive, but that the changes generally are in the direction predicted by the theory of Burbaum, Raines, Albery & Knowles [(1989) Biochemistry 28, 9283-9305] for an enzyme catalysing a thermodynamically irreversible reaction. 7. Michaelis-Menten parameters for the hydrolysis of six beta-D-galactopyranosylpyridinium ions and ten aryl beta-galactosides by ebgab were measured. 8. The derived beta 1g values are the same as those for ebgb (which has only the Trp-977----Cys change) and significantly different from those for ebgo (the wild-type enzyme) and ebga. 9. The alpha- and beta-deuterium secondary isotope effects on the hydrolysis of the galactosyl-enzyme of 1.08 and 1.00 are difficult to reconcile with the pyranose ring in this intermediate being in the 4C1 conformation.

SUBMITTER: Elliott AC

PROVIDER: S-EPMC1130902 | biostudies-other | 1992 Feb

REPOSITORIES: biostudies-other

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Project description:The kinetics of hydrolysis of a series of synthetic substrates by two experimentally evolved forms ('evolvants'), ebgabcd and ebgabcde, of the second beta-galactosidase of Escherichia coli have been measured. The ebgabcd enzyme differs from the wild-type (ebgo) enzyme by Asp92-->Asn (a) and Trp977-->Cys (b) changes in the large subunit, as well as two changes hitherto considered to have no kinetic effect, Ser979-->Gly in the large subunit (c) and Glu122-->Gly in the small subunit (d). The enzyme ebgabcde contains in addition a Glu93-->Lys change in the large subunit (e). Comparison of ebgabcd with ebgab [Elliott, K, Sinnott, Smith, Bommuswamy, Guo, Hall and Zhang (1992) Biochem. J. 282, 155-164] indicates that the c and d changes in fact accelerate the hydrolysis of the glycosyl-enzyme intermediate by a factor of 2.5, and also decrease the charge on the aglycone oxygen atom at the first transition state; the charge on the glycone, however, is unaltered [see K, Konstantinidis, Sinnott and Hall (1993) Biochem. J. 291, 15-17]. The e mutation causes a fall in the degalactosylation rate of about a factor of 3, and its occurrence only together with c and d mutations [Hall, Betts and Wootton (1989) Genetics 123, 635-648] suggests that degalactosylation of a hypothetical ebgabe enzyme would be so slow that the enzyme would have no biological advantage over the ancestral ebgab. The transfer products from galactosyl-ebgabcd and galactosyl-ebgabcde to high concentrations to glucose have been measured; the predominant product is allolactose, but significant quantities of lactose are also formed; however, at apparent kinetic saturation of the galactosyl-enzyme, hydrolysis rather than transfer is the preponderant pathway. A knowledge of the rates of enzyme-catalysed exchange of 18O from [1-18O]galactose to water permits the construction of the free-energy profiles for hydrolysis of lactose by begabcd and ebgabcde. As with the other evolvants, changes in the profile away from the rate-determining transition state are essentially random, and there is no correlation between the changes in the free energies of intermediates and of their flanking transition states. We consider the aggregate of our kinetic data on the ebg system to be telling experimental support for the theoretical objections of Pettersson [Pettersson (1992) Eur. J. Biochem. 206, 289-295 and previous papers] to the Albery-Knowles theory of the evolution of enzyme kinetic activity.

Project description:Plasmids containing the ebgAo and ebgAa genes of Escherichia coli under the control of the lac repressor and promoter have been constructed and inserted into Salmonella typhimurium CH3. This system expresses the large subunit of the ebgo and ebga beta-galactosidase in high yield (20-60% of total protein). The large subunits have been purified to homogeneity. As isolated they are tetramers of significant catalytic activity; the N-terminal amino acid residue is Met, but it is not formylated. The kcat. values for a series of aryl galactosides were 6-200-fold reduced from the corresponding values for the holoenzymes. kcat/Km Values for glycosides of acidic aglycones, though, were unchanged, whilst kcat./Km values for galactosides of less acidic aglycones showed a modest (up to 10-fold) decrease. The kcat. values for glycosides of acidic aglycones hydrolysed by ebgo and ebga large subunits were essentially invariant with aglycone pK, suggesting that hydrolysis of the galactosyl-enzyme intermediate had become rate-determining for these substrates. Rate-determining hydrolysis of the glycosyl-enzyme intermediate was confirmed by pre-steady-state measurements and nucleophilic competition with methanol. Absence of the small subunit was thus estimated to cause a 200-fold decrease in degalactosylation rate for ebgo and a 20-fold one for ebga. beta 1g(V/K) values of -0.57 +/- 0.08 for ebgo and -0.54 +/- 0.08 for ebga isolated subunits were significantly more negative than for holoenzymes. It is suggested that the small subunit is associated with the optimal positioning of the electrophilic Mg2+ ions in these enzymes. Use of PCR in the construction of the plasmid also inadvertently led to the production of psi ebgo large subunit in which there was a PCR-introduced Leu9-->His change. Values of kcat. for aryl galactosides, calculated on the assumption that the psi ebgo large subunit, like the ebgo and ebga large subunits, was 100% active as isolated, were about an order of magnitude lower than for true ebgo large subunit, whilst Km values were similar. The very significant kinetic effect of this inadvertant site-undirected mutagenesis indicates that quite large kinetic effects of amino-acid replacements in enzymes may have no obvious mechanistic significance.

Project description:BACKGROUND:Photosynthetic microorganisms that directly channel solar energy to the production of molecular hydrogen are a potential future biofuel system. Building such a system requires installation of a hydrogenase in the photosynthetic organism that is both tolerant to oxygen and capable of hydrogen production. Toward this end, we have identified the [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii "Deep ecotype" that is able to be heterologously expressed in cyanobacteria and has tolerance to partial oxygen. The A. macleodii enzyme shares sequence similarity with the uptake hydrogenases that favor hydrogen uptake activity over hydrogen evolution. To improve hydrogen evolution from the A. macleodii hydrogenase, we examined the three Fe-S clusters found in the small subunit of many [NiFe] uptake hydrogenases that presumably act as a molecular wire to guide electrons to or from the active site of the enzyme. Studies by others altering the medial cluster of a Desulfovibrio fructosovorans hydrogenase from 3Fe-4S to 4Fe-4S resulted in two-fold improved hydrogen evolution activity. RESULTS:We adopted a strategy of screening for improved hydrogenase constructs using an Escherichia coli expression system before testing in slower growing cyanobacteria. From the A. macleodii enzyme, we created a mutation in the gene encoding the hydrogenase small subunit that in other systems is known to convert the 3Fe-4S medial cluster to 4Fe-4S. The medial cluster substitution did not improve the hydrogen evolution activity of our hydrogenase. However, modifying both the medial cluster and the ligation of the distal Fe-S cluster improved in vitro hydrogen evolution activity relative to the wild type hydrogenase by three- to four-fold. Other properties of the enzyme including thermostability and tolerance to partial oxygen did not appear to be affected by the substitutions. CONCLUSIONS:Our results show that substitution of amino acids altering the ligation of Fe-S clusters in the A. macleodii [NiFe] uptake hydrogenase resulted in increased hydrogen evolution activity. This activity can be recapitulated in multiple host systems and with purified protein. These results validate the approach of using an E. coli-cyanobacteria shuttle system for enzyme expression and improvement.

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The catalytic consequences of experimental evolution. Studies on the subunit structure of the second (ebg) beta-galactosidase of Escherichia coli, and on catalysis by ebgab, an experimental evolvant containing two amino acid substitutions.

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