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At least 541 records · Page 30Linked to original sources

PubMed2005

Production of beta-galactosidase by Bifidobacteria as influenced by various culture conditions.

Beta-Galactosidase production by Bifidobacterium longum CCRC 15708, Bifidobacterium longum B6 and Bifidobacterium infantis CCRC 14633 was first examined with B. longum CCRC 15708 showing the highest production of beta-galactosidase and the highest specific activity. Further study with B. longum CCRC 15708 revealed that the highest level of beta-galactosidase was produced with lactose and yeast extract as carbon and nitrogen sources, respectively. Optimal enzyme production occurred at an initial pH of 6.5 and at 37 degrees C. Under these optimum culture conditions, a maximumbeta-galactosidase activity of 18.6 U/ml could be obtained after 16 h of fermentation in a medium contain 4% lactose, 3.5% yeast extract, 0.3% K2HPO4, 0.1% KH2PO4, 0.05% MgSO4.7H2O and 0.03% L-cysteine. The highest transgalactosylation activity was also detected in this culture after 14-16 h of fermentation.

Bifidobacterium↗

PubMed2004

Engineered autolytic yeast strains secreting Kluyveromyces lactis beta-galactosidase for production of heterologous proteins in lactose media.

Secretion of the heterologous Kluyveromyces lactis beta-galactosidase into culture medium by several Saccharomyces cerevisiae osmotic-remedial thermosensitive-autolytic mutants was assayed and proved that new metabolic abilities were conferred since the constructed strains were able to grow in lactose-containing media. Cell growth became independent of a lactose-uptake mechanism. Higher levels of extra-cellular and intra-cellular beta-galactosidase production, lactose consumption and growth were obtained with the LHDP1 strain, showing a thermosensitive-autolytic phenotype as well as being peptidase-defective. The recombinant strain LHDP1 presented the highest beta-galactosidase yields from biomass and the lowest ethanol levels from lactose. This strain is effective for the heterologous production and release of K. lactis beta-galactosidase into the extra-cellular medium after osmotic shock.

Cheese↗

PubMed2006

Induction and characterization of an unusual alpha-D-galactosidase from Talaromyces flavus.

An extracellular alpha-d-galactosidase from Talaromyces flavus CCF 2686 with extremely broad and unusual acceptor specificity is produced exclusively in the presence of the specific inducer--6-deoxy-D-glucose (quinovose). The procedure for the preparation of this very expensive substance has been modified and optimized. Surprisingly, any of other common alpha-D-galactosidase inducers or substrates, e.g., D-galactose, melibiose and raffinose, did not stimulate its production. The crude alpha-D-galactosidase preparation was purified by anion-exchange chromatography and three isoenzymes with different substrate specificities were identified. The main isoenzyme (alphaGal1) was further purified by cation-exchange chromatography and fully characterized. When compared with other alpha-galactosidases and also with other isoenzymes produced by T. flavus, it showed a markedly different regioselectivity and also negligible hydrolytic activity towards melibiose. Moreover, it was active on polymeric substrates (locust bean gum, guar gum) and significantly inhibited by alpha-D-galactopyranosyl azide, D-galactose, D-xylose, melibiose, methyl alpha- and beta-D-galactopyranoside and lactose.

Deoxyglucose↗

PubMed2005

Cold-active beta-galactosidase from Arthrobacter sp. C2-2 forms compact 660 kDa hexamers: crystal structure at 1.9A resolution.

The X-ray structure of cold-active beta-galactosidase (isoenzyme C-2-2-1) from an Antarctic bacterium Arthrobacter sp. C2-2 was solved at 1.9A resolution. The enzyme forms 660 kDa hexamers with active sites opened to the central cavity of the hexamer and connected by eight channels with exterior solvent. To our best knowledge, this is the first cold-active beta-galactosidase with known structure and also the first known beta-galactosidase structure in the form of compact hexamers. The hexamer organization regulates access of substrates and ligands to six active sites and this unique packing, present also in solution, raises questions about its purpose and function. This enzyme belongs to glycosyl hydrolase family 2, similarly to Escherichia coli beta-galactosidase, forming tetramers necessary for its enzymatic function. However, we discovered significant differences between these two enzymes affecting the ability of tetramer/hexamer formation and complementation of the active site. This structure reveals new insights into the cold-adaptation mechanisms of enzymatic pathways of extremophiles.

Amino Acid Sequence↗

PubMed2005

Cloning, expression, and purification of a recombinant cold-adapted beta-galactosidase from antarctic bacterium Pseudoalteromonas sp. 22b.

The gram-negative antarctic bacterium Pseudoalteromonas sp. 22b, isolated from the alimentary tract of krill Thyssanoessa macrura, synthesizes an intracellular cold-adapted beta-galactosidase. The gene encoding this beta-galactosidase has been PCR amplified, cloned, expressed in Escherichia coli, purified, and characterized. The enzyme is active as a homotetrameric protein, and each monomer consists of 1028 amino acid residues. The enzyme was purified to homogeneity (50% recovery of activity) by using the fast, two-step procedure, including affinity chromatography on PABTG-Sepharose. Enzymatic properties of the recombinant protein are identical to those of native Pseudoalteromonas sp. 22b beta-galactosidase. The enzyme is cold-adapted and at 10 degrees C retains 20% of maximum activity. The purified enzyme displayed maximum activity close to 40 degrees C and at pH of 6.0-8.0. PNPG was its preferred substrate (58% higher activity than against ONPG). The enzyme was particularly thermolabile, losing all activities within 10 min at 50 degrees C. The hydrolysis of lactose in a milk assay revealed that 90% of milk lactose was hydrolyzed during 6 h at 30 degrees C and during 28 h at 15 degrees C. Because of its attributes, the recombinant Pseudoalteromonas sp. 22b beta-galactosidase could be applied at refrigeration temperatures for production of lactose-reduced dairy products.

Amino Acid Sequence↗

PubMed2006

Purification of a beta-galactosidase from cotyledons of Hymenaea courbaril L. (Leguminosae). Enzyme properties and biological function.

Beta-galactosidases are enzymes that can be found in most living beings and in the plant kingdom its activity and genes have been detected in several tissues such as ripening fruits, developing leaves and flowers and storage tissues such as cotyledons. In plants, their activities are usually associated with the secondary metabolism or with oligosaccharide or polysaccharide degradation. Polysaccharide specific beta-galactosidases include beta-galactanases, which attack pectic polymers and beta-galactosidases that attack xyloglucans (XG). In the present work we purified an XG-specific beta-galactosidase (named hcbetagal) from cotyledons of developing seedlings of Hymenaea courbaril, a legume tree from the Neotropical region of the world. The enzyme has a molecular weight of 52-62 kDa and was shown to attack specifically xyloglucan oligosaccharides (XGOs) but not the polymer. It has a pH optimum between 3 and 4 and at this pH range the enzyme increases activity linearly up to 50 degrees C. Kinetic studies showed that hcbetagal is inhibited competitively by free galactose (K(i) = 3.7). The biochemical properties of hcbetagal as a whole suggest that it is involved in storage xyloglucan mobilisation during seedling development. Its high specificity towards XGOs, the low pH optimum and the fact that it is inhibited by its product (galactose) suggest that hcbetagal might be one of the biochemical control points in xyloglucan catabolism in vivo. A possible relationship with functional stability of the wall during cell death as cotyledons undergo senescence is discussed.

Cell Death↗

PubMed1991

A bireactant, irreversible, active-site-directed inhibitor of beta-D-galactosidase (Escherichia coli). Synthesis and properties of (1/2,5,6)-2-(3-azibutylthio)-5,6-epoxy-3-cyclohexen-1-ol.

(1/2,5,6)-2-(3-Azibutylthio)-5,6-epoxy-3-cyclohexen-1-ol (1) was synthesized and was found to irreversibly inactivate beta-D-galactosidase (Escherichia coli). The inactivation was prevented by the presence of isopropyl 1-thio-beta-D-galactopyranoside (IPTG). The vinyloxirane group of 1 reacted with water and other nucleophiles, especially at higher pH values. Reaction of 1 with beta-D-galactosidase was slow enough so that a competitive-inhibition constant (Ki) of 29mM could be determined. The inhibition constant for (1,2/3,6)-6-(3-azibutylthio)-2-bromo-4-cyclohexene-1,3-diol (2), the precursor of the bireactant inhibitor 1, was 13 mM, while that of (1,3/2,4)-3-(3-azibutylthio)-5-cyclohexene-1,2,4-triol (3), the product formed when the reactant is allowed to react with water, was 23mM. After irradiation by light, beta-D-galactosidase that had initially been treated with the bireactant compound and then digested with trypsin, showed a new pattern of elution from h.p.l.c., indicating that there was reaction at two regions of the beta-D-galactosidase molecule.

Azo Compounds↗

PubMed2002

1-O-Acetyl-beta-D-galactopyranose: a novel substrate for the transglycosylation reaction catalyzed by the beta-galactosidase from Penicillium sp.

1-O-Acetyl-beta-D-galactopyranose (AcGal), a new substrate for beta-galactosidase, was synthesized in a stereoselective manner by the trichloroacetimidate procedure. Kinetic parameters (K(M) and k(cat)) for the hydrolysis of 1-O-acetyl-beta-D-galactopyranose catalyzed by the beta-D-galactosidase from Penicillium sp. were compared with similar characteristics for a number of natural and synthetic substrates. The value for k(cat) in the hydrolysis of AcGal was three orders of magnitude greater than for other known substrates. The beta-galactosidase hydrolyzes AcGal with retention of anomeric configuration. The transglycosylation activity of the beta-D-galactosidase in the reaction of AcGal and methyl beta-D-galactopyranoside (1) as substrates was investigated by 1H NMR spectroscopy and HPLC techniques. The transglycosylation product using AcGal as a substrate was beta-D-galactopyranosyl-(1-->6)-1-O-acetyl-beta-D-galactopyranose (with a yield of approximately 70%). In the case of 1 as a substrate, the main transglycosylation product was methyl beta-D-galactopyranosyl-(1-->6)-beta-D-galactopyranoside. Methyl beta-D-galactopyranosyl-(1-->3)-beta-D-galactopyranoside was found to be minor product in the latter reaction.

Galactosides↗

PubMed1999

Biochemical studies on leukocyte and fibroblast human beta-galactosidase.

OBJECTIVES: Some biochemical characteristics of the human leukocyte and fibroblast beta-galactosidase were studied. DESIGN AND METHODS: Leukocyte and fibroblast enzyme activity was determined fluorometricaly using 4-methylumbelliferyl-beta-D-galactoside as artificial substrate. Optimum pH, Km, Vmax and thermostability of the enzyme at 42 degrees C were determined. RESULTS: The leukocyte and fibroblast enzyme has an optimum pH at 4.2, which is in agreement with the lysosomal origin of the enzyme. The Km of the enzyme was 0.62 in leukocytes and 0.67 in fibroblasts, and Vmax was 289.9 nmol/h/mg of protein and 1779.2 nmol/h/mg of protein in the two tissues, respectively. When fibroblast or leukocyte beta-galactosidase was pre-incubated at 42 degrees C, it did not retain its activity because the residual activity after 80 minutes of pre-incubation at this temperature was lower than 30% of the initial activity both in leukocytes and fibroblasts. CONCLUSIONS: This was the first study of Km, Vmax and thermostability of beta-galactosidase performed on leukocytes and provided data for a better characterization of the enzyme beta-galactosidase, allowing the improvement of the analytical conditions.

Fibroblasts↗

PubMed2001

Characteristics of an alpha-galactosidase associated with grape flesh.

alpha-galactosidase activity in grape flesh (Vitis venifera L. Muscat of Alexandria) was characterized by a marked increase in its activity 4 weeks after fruit bearing. After 12 weeks the specific activity of the enzyme had increased 15-fold. Several other glycosidases were measured at different stages of fruit development but none showed the increased levels of activity displayed by this alpha-galactosidase. alpha-Galactosidase activity (unit/g.fresh wt) increased by 52% during postharvest storage, whereas the unripe grape showed a "stagnancy" for 10-15 days prior to the increase. An alpha-galactosidase was partially purified ca. 103-fold from grape flesh of Vitis labruscana Honey black, by a procedure involving ammonium sulfate fractionation, Biogel P-60, melibiose-agarose, and Sephacryl S-200 chromatographic separations. The enzyme was effectively separated by affinity chromatography on melibiose-agarose, and was a monomer of 40-45 kDa as determined by SDS-PAGE and Sephacryl S-200 chromatographic analysis. The hydrolysis rate of p-nitrophenyl-alpha-D-Gal (PNP-alpha-D-Gal) was 4.2 times higher than that of PNP-beta-D-Gal, implying an apparent alpha-anomer specificity, and natural oligosaccharides such as melibiose, stachyose, and raffinose were also considerably hydrolyzed. The enzyme was active over a narrow pH range with an optimal hydrolysis of stachyose and PNP-alpha-D-Gal at pH 6.0 and 7.0, respectively. EDTA or 1,10-phenanthroline did not substantially affect enzyme activity.

Chromatography, Gel↗

PubMed2002

Expression of beta-galactosidase by recombinant respiratory syncytial viruses for microneutralization assay.

The beta-galactosidase gene (lacZ) was inserted into a recombinant respiratory syncytial virus (RSV) A2 strain of subgroup A RSV (designated as A-lacZ) and a chimeric RSV that had the G and F surface glycoproteins of A2 replaced by those of the subgroup B RSV 9320 strain (designated as B-lacZ). Both recombinant RSVs, A-lacZ and B-lacZ, grew well in tissue culture and expressed high levels of beta-galactosidase. Using these two beta-galactosidase-expressing recombinant RSVs, a novel microneutralization assay was developed to measure serum anti-RSV neutralizing antibody from subgroup A or subgroup B RSV infection. The assay was carried out in 96-well plates and the unneutralized virus was quantitated by spectrophotometric measurement of the beta-galactosidase enzymatic reaction following incubation of the infected cell lysate with the enzyme substrate, chlorophenol red beta-D-galactopyranoside (CPRG). Adult human sera positive for anti-RSV antibody as shown by Western blot analysis and subgroup A or subgroup B RSV infected monkey sera were examined for the levels of anti-RSV neutralizing antibodies by the microneutralization assay in comparison with the plaque reduction neutralization assay. A higher antibody titer was detected when the neutralization assay was performed with the homologous RSV than the heterologous RSV, indicating that neutralization assay could distinguish antigenic differences between the two RSV subgroups. The microneutralization assay is comparable to the plaque reduction neutralization assay in sensitivity, but it is rapid, less laborious and suitable for screening a large number of samples.

Adult↗

PubMed2000

Candida glabrata shuttle vectors suitable for translational fusions to lacZ and use of beta-galactosidase as a reporter of gene expression.

The functionality of beta-galactosidase encoded by the E. coli lacZ gene as a reporter of gene expression in C. glabrata was investigated. C. glabrata/E. coli shuttle vectors were constructed, containing both a C. glabrata CEN-ARS cassette, to allow regular segregation and episomal replication of the plasmids, and the lacZ coding sequence of E. coli. The functionality of beta-galactosidase in C. glabrata was verified by inserting the promoter and the 5' coding region of the HIS3 gene from C. glabrata directionally upstream of the lacZ gene. By fusing the promoter of the copper-controlled MTII gene to the lacZ reporter, we showed that beta-galactosidase activity can be differentially induced in C. glabrata. beta-galactosidase reporter activities were detected qualitatively by an indirect filter assay and quantitatively from permeabilized cells.

Candida↗

PubMed1998

Cloning, functional expression and purification of endo-beta-galactosidase from Flavobacterium keratolyticus.

Endo-beta-galactosidase (EC 3.2.1.103) is an enzyme that hydrolyzes internal endo-beta-galactosyl linkages in keratan sulfate, and glycoconjugates with N-acetyl-lactosamine repeating units. Here, we report the cloning of the endo-beta-galactosidase-encoding gene from Flavobacterium keratolyticus, its expression in Escherichia coli and the purification of the enzyme. The enzyme was purified over 15000-fold to apparent homogeneity. The purified endo-beta-galactosidase consists of a single band of about 43kDa on SDS-PAGE and has a specific activity of 148micro/mg. Based on peptide sequences derived from the purified enzyme, a full-length clone encoding endo-beta-galactosidase was isolated from F. keratolyticus genomic DNA. The gene contains a single open reading frame coding for a protein of 422 amino acid residues with a putative N-terminal signal peptide. Its authenticity was confirmed by colinearity of deduced amino acid sequences with the peptide sequences, and synthesis of enzyme in E. coli.

Amino Acid Sequence↗

PubMed1997

Molecular form and subcellular distribution of acid beta-galactosidase in fibroblasts from patients with GM1 gangliosidosis, Morquio B disease and galactosialidosis.

The molecular form and subcellular distribution of acid beta-galactosidase in cultured fibroblasts from patients with beta-galactosidase deficiency (GM1-gangliosidosis, Morquio B disease and galactosialidosis) were studied, using antibodies against three different forms of the human enzyme: a high-molecular-weight multienzymic complex, a recombinant 84-kDa precursor, and a 64-kDa tryptic product of the precursor. The mature enzyme from normal fibroblasts was immunoprecipitated by the anti-complex and anti-64-kDa protein antibodies, but not by the anti-84-kDa precursor one. immunofluorescence staining of normal fibroblasts revealed the granular (lysosomal) distribution with anti-64-kDa protein antibody and the perinuclear reticular distribution with anti-84-kDa precursor antibody, probably representing the Golgi apparatus. Both patterns were demonstrated in Morquio B disease, but the residual enzyme activity was exclusively due to the mature enzyme. In Type 1 galactosialidosis, most of the expressed enzyme was detected as the precursor form with a perinuclear reticular distribution. In type 2 galactosialidosis, more than half of the enzyme activity was due to the mature form with a lysosomal distribution. Fibroblasts from a patient with GM1 gangliosidosis, expressing no beta-galactosidase mRNA, did not react against either anti-64-kDa protein antibody or anti-84-kDa precursor antibody. The combined use of immunoprecipitation and immunostaining was useful for analysing the pathophysiology of the intracellular processing and transport of the mutant beta-galactosidase.

Antibody Specificity↗

PubMed1998

Properties of an alpha-galactosidase, and structure of its gene galA, within an alpha-and beta-galactoside utilization gene cluster of the hyperthermophilic bacterium Thermotoga maritima.

Thermotoga maritima represents one of the few hyperthermophilic bacteria currently known. The chromosomal alpha-galactosidase gene of T. maritima strain MSB8 has been cloned and its nucleotide sequence was determined. The gene, designated galA, has coding capacity for a 552 residue polypeptide with a calculated molecular mass of 63,653 Da. GalA was found to be flanked by other genes probably involved in galactoside breakdown and utilization. The previously sequenced beta-galactosidase gene, lacZ, is localized immediately upstream of galA while two open reading frames that putatively encode enzymes of galactose catabolism, i.e. galactose-1-phosphate uridylytransferase (galT) and galactokinase (galK), were found downstream of galA. The identified genes are extremely close together or even overlap and have the same orientation, so they could all be part of one galactoside utilization operon of T. maritima MSB8. GalA displayed low-level amino acid sequence similarity with alpha-galactoside of glycosyl hydrolase family 36. However, GalA is smaller than the other members of this enzyme family. The galA gene was expressed in Escherichia coli and the recombinant alpha-galactosidase was purified and characterized. The molecular mass of the recombinant enzyme was estimated at about 62 kDa by denaturting gel electrophoresis. Maximal hydrolysis of the chromogenic substrate p-nitrophenyl-alpha-D-galactopyranoside was measured at pH 5.0-5.5 and 90-95 degrees C (5 min assay). Divalent cations were not required for activity. The enzyme released galactose from raffinose, melibiose and the synthetic substrates p-nitrophenyl-and omicron-nitrophenyl-alpha-D-galactopyranoside. The T. maritima alpha-galactosidase thus was highly specific for the galactose moiety and the alpha-anomeric configuration of the glycosidic linkage. Its extreme thermal stability (t 1/2 = 6.5 h at 85 degrees C) makes this enzyme an interesting candidate for biotechnological applications.

Amino Acid Sequence↗

PubMed1991

Inactivation mechanism of tetrameric beta-galactosidase by gamma-rays involves both fragmentation and temperature-dependent denaturation of protomers.

The radiation inactivation method is widely used to estimate the molecular size of membrane-bound enzymes, receptors, and transport systems in situ. The method is based on the principle that exposure of frozen solutions or lyophilized protein preparations to increasing doses of ionizing radiations results in a first-order decay of biological activity proportional to radiation inactivation size of the protein. This parameter is believed to reflect the "functional unit" of the protein defined as the minimal assembly of structure (protomers) required for expression of a given biological activity. We tested the functional unit as a concept to interpret radiation inactivation data of proteins with Escherichia coli beta-galactosidase, where the protomers are active only when associated in a tetramer. Gamma-Irradiation of beta-galactosidase at both -78 and 38 degrees C followed by quantitation of the residual unfragmented promoter band by SDS-polyacrylamide gel electrophoresis yielded the protomer size, indicating that only one protomer is fragmented by each radiation hit. By following the enzyme activity as a function of dose it was found that only the protomer that has been directly hit and fragmented at -78 degrees C was effectively inactivated. In contrast, at 38 degrees C, it was the whole tetramer that was inactivated. beta-Galactosidase cannot have two different functional units depending on temperature. The inactivation of the whole beta-galactosidase tetramer at 38 degrees C is in fact related to protomer fragmentation but also to the production of stable denatured protomers (detected by gel-filtration HPLC and differential UV spectroscopy) due to energy transfer from fragmented protomers toward unhit protomers.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme Activation↗

PubMed1977

Escherichia coli DNA-directed beta-galactosidase synthesis in presence and absence of Ca2+.

DNA-dependent synthesis of beta-galactosidase was optimized in extracts made from cells lysed by a standard French pressure cell. Extracts made at 3200 psi synthesized up to 25-fold more beta-galactosidase than extracts made at 7500 psi. beta-Galactosidase synthesis was cyclic 3', 5' AMP dependent, as expected, and in optimal conditions transcription and translation proceeded at 8.6 nucleotides and 2.7 amino acids per s, respectively. The high pressure extracts were stimulated 3- to 5-fold by Ca2+, especially at low Mg2+ concentrations. In contrast, extracts prepared at low pressure were inhibited as much as 50-fold by Ca2+ ions. The inhibition by Ca2+ was analyzed further. Addition of kasugamycin, an antibiotic that acts on ribosomes, to reactions containing Ca2+ stimulated beta-galactosidase synthesis to nearly control levels. Extracts from a kasugamycin resistant mutant were neither inhibited by Ca2+ nor stimulated by the addition of kasugamycin to in vitro reactions containing Ca2+. The change in the mutant was ascribed to the ribosomes by testing combinations of soluble proteins, ribosome wash, and ribosomes from parental and mutant strains. These results suggest that Ca2+ ions inhibit translation by ribosomes, very likely at an initiation step; and that they enhance enzyme synthesis only in conditions where translation is inefficient (high-pressure extracts at low concentrations of Mg2+, for example). This latter effect is probably a consequence of increased RNA stability in the presence of Ca2+ (Cremer, K., and Schlessinger, D. (1974), J. Biol. Chem. 249,4730).

Aminoglycosides↗

PubMed1976

beta-Galactosidase alpha complementation: properties of the complemented enzyme and mechanism of the complementation reaction.

Intracistronic alpha complementation involving Escherichia coli beta-galactosidase occurs between the cyanogen bromide peptide CB2, derived from residues 3-92 of beta-galactosidase (Langley, K.E., Fowler, A.V., and Zabin, I. (1975), J. Biol. Chem. 250, 2587), and the defective beta-galactosidase from the Z-deletion mutant strain M15. The M15 protein, a dimer, lacks residues 11-41 of beta-galactosidase (Langley, K.E., Villarejo, M.R., Fowler, A.V., Zamenhof, P.J., and Zabin, I. (1975), Proc. Natl. Acad. Sci. U.S.A. 72, 1254). The complemented enzyme formed from purified components has a molecular weight of 533 000+/-25 000, is therefore tetrameric, and has a probable stoichiometry of 1 CB2:1 M15 monomer. The complemented enzyme has the same Km for substrate as wild type enzyme, but is less stable to heat or urea treatment. The overall equilibrium constant for the complementation reaction is approximately 1-2 X 10(9) M-1. Initial velocity studies indicate saturation kinetics when either component is fixed and limiting, with an apparent Kd of about 10(-6) M. A first-order rate constant of 0.05-0.1 min-1 was estimated. The kinetics favor a model of rapid complex formation, followed by slow conformational change, as the mechanism of activation. Ultraviolet difference spectroscopy indicated an increased absorbance in the 290-300 nm region as a result of the complementation reaction. The kinetics of the increase suggest that two processes, one rapid and the other slower, could be responsible. The temperature dependence of complementation (Ea approximately 24 000 cal) is also consistent with the rate-determining step being a conformational change.

Bacterial Proteins↗