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Catalytic consequences of experimental evolution: catalysis by a 'third-generation' evolvant of the second beta-galactosidase of Escherichia coli, ebgabcde, and by ebgabcd, a 'second-generation' evolvant containing two supposedly 'kinetically silent' mutations.

ABSTRACT: 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.

SUBMITTER: Krishnan S

PROVIDER: S-EPMC1136208 | biostudies-other | 1995 Dec

REPOSITORIES: biostudies-other

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Project description: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.

Project description:Eimeria is a common genus of apicomplexan parasites that infect diverse vertebrates, most notably poultry, causing serious disease and economic losses. Eimeria species have complex life-cycles consisting of three developmental stages. However, the molecular basis of the Eimeria reproductive mode switch remains an enigma.Total RNA extracted from second- (MZ-2) and third-generation merozoites (MZ-3) of Eimeria necatrix was subjected to transcriptome analysis using RNA sequencing (RNA-seq) followed by qRT-PCR validation.A total of 6977 and 6901 unigenes were obtained from MZ-2 and MZ-3, respectively. Approximately 2053 genes were differentially expressed genes (DEGs) between MZ-2 and MZ-3. Compared with MZ-2, 837 genes were upregulated and 1216 genes were downregulated in MZ-3. Approximately 95 genes in MZ-2 and 48 genes in MZ-3 were further identified to have stage-specific expression. Gene ontology category and KEGG analysis suggested that 216 upregulated genes in MZ-2 were annotated by 70 GO assignments, 242 upregulated genes were associated with 188 signal pathways, while 321 upregulated genes in MZ-3 were annotated by 56 GO assignments, 322 upregulated genes were associated with 168 signal pathways. The molecular functions of upregulated genes in MZ-2 were mainly enriched for protein degradation and amino acid metabolism. The molecular functions of upregulated genes in MZ-3 were mainly enriched for transcriptional activity, cell proliferation and cell differentiation.To the best of our knowledge, this is the first RNA-seq data study of the MZ-2 and MZ-3 stages of E. necatrix; it demonstrates a high number of differentially expressed genes between the MZ-2 and MZ-3 of E. necatrix. This study forms a basis for deciphering the molecular mechanisms underlying the shift from the second to third generation schizogony in Eimeria. It also provides valuable resources for future studies on Eimeria, and provides insight into the understanding of reproductive mode plasticity in different Eimeria species.

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Catalytic consequences of experimental evolution: catalysis by a 'third-generation' evolvant of the second beta-galactosidase of Escherichia coli, ebgabcde, and by ebgabcd, a 'second-generation' evolvant containing two supposedly 'kinetically silent' mutations.

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