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PubMed1996

Three alpha-galactosidase genes of Trichoderma reesei cloned by expression in yeast.

Three alpha-galactosidase genes, agl1, agl2 and agl3, were isolated from a cDNA expression library of Trichoderma reesei RutC-30 constructed in the yeast Saccharomyces cerevisiae by screening the library on plates containing the substrate 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside. The genes agl1, agl2 and agl3 encode 444, 746 and 624 amino acids, respectively, including the signal sequences. The deduced amino acid sequences of AGLI and AGLIII showed similarity with the alpha-galactosidases of plant, animal, yeast and filamentous fungal origin classified into family 27 of glycosyl hydrolases whereas the deduced amino acid sequence of AGLII showed similarity with the bacterial alpha-galactosidases of family 36. The enzymes produced by yeast were analysed for enzymatic activity against different substrates. AGLI, AGLII and AGLIII were able to hydrolyse the synthetic substrate p-nitrophenyl-alpha-D-galactopyranoside and the small galactose-containing oligosaccharides, melibiose and raffinose. They liberated galactose from polymeric galacto(gluco)mannan with different efficiencies. The action of AGLI towards polymeric substrates was enhanced by the presence of the endo-1,4-beta-mannanase of T. reesei. AGLII and AGLIII showed synergy in galacto(gluco)mannan hydrolysis with the endo-1,4-beta-mannanase of T. reesei and a beta-mannosidase of Aspergillus niger. The calculated molecular mass and the hydrolytic properties of AGLI indicate that it corresponds to the alpha-galactosidase previously purified from T. reesei.

Amino Acid Sequence↗
PubMed2007

Characterization and molecular cloning of a heterodimeric beta-galactosidase from the probiotic strain Lactobacillus acidophilus R22.

Beta-galactosidase from the probiotic strain Lactobacillus acidophilus R22 was purified to apparent homogeneity by ammonium sulphate fractionation, hydrophobic interaction, and affinity chromatography. The enzyme is a heterodimer consisting of two subunits of 35 and 72 kDa, as determined by gel electrophoresis. The optimum temperature of beta-galactosidase activity was 55 degrees C (10-min assay) and the range of pH 6.5-8, respectively, for both o-nitrophenyl-beta-D-galactopyranoside (oNPG) and lactose hydrolysis. The Km and Vmax values for lactose and oNPG were 4.04+/-0.26 mM, 28.8+/-0.2 micromol D-glucose released per min per mg protein, and 0.73+/-0.07 mM, 361+/-12 micromol o-nitrophenol released per min per mg protein, respectively. The enzyme was inhibited by high concentrations of oNPG with Ki,s=31.7+/-3.5 mM. The enzyme showed no specific requirements for metal ions, with the exception of Mg2+, which enhanced both activity and stability. The genes encoding this heterodimeric enzyme, lacL and lacM, were cloned, and compared with other beta-galactosidases from lactobacilli. Beta-galactosidase from L. acidophilus was used for the synthesis of prebiotic galacto-oligosaccharides (GOS) from lactose, with the maximum GOS yield of 38.5% of total sugars at about 75% lactose conversion.

Bacterial Proteins↗
PubMed2006

Bioinformatic, genetic, and biochemical evidence that some glycoside hydrolase family 42 beta-galactosidases are arabinogalactan type I oligomer hydrolases.

Glycoside hydrolases are organized into glycoside hydrolase families (GHFs) and within this larger group, the beta-galactosidases are members of four families: 1, 2, 35, and 42. Most genes encoding GHF 42 enzymes are from prokaryotes unlikely to encounter lactose, suggesting a different substrate for these enzymes. In search of this substrate, we analyzed genes neighboring GHF 42 genes in databases and detected an arrangement implying that these enzymes might hydrolyze oligosaccharides released by GHF 53 enzymes from arabinogalactan type I, a pectic plant polysaccharide. Because Bacillus subtilis has adjacent GHF 42 and GHF 53 genes, we used it to test the hypothesis that a GHF 42 enzyme (LacA) could act on the oligosaccharides released by a GHF 53 enzyme (GalA) from galactan. We cloned these genes, plus a second GHF 42 gene from B. subtilis, yesZ, into Escherichia coli and demonstrated that cells expressing LacA with GalA gained the ability to use galactan as a carbon source. We constructed B. subtilis mutants and showed that the increased beta-galactosidase activity generated in response to the addition of galactan was eliminated by inactivating lacA or galA but unaffected by the inactivation of yesZ. As further demonstration, we overexpressed the LacA and GalA proteins in E. coli and demonstrated that these enzymes degrade galactan in vitro as assayed by thin-layer chromatography. Our work provides the first in vivo evidence for a function of some GHF 42 beta-galactosidases. Similar functions for other beta-galactosidases in both GHFs 2 and 42 are suggested by genomic data.

Bacillus subtilis↗
PubMed1990

Purification and characterization of thermostable beta-mannanase and alpha-galactosidase from Bacillus stearothermophilus.

Bacillus stearothermophilus secretes beta-mannanase and alpha-galactosidase enzymatic activities capable of hydrolyzing galactomannan substrates. Expression of the hemicellulase activities in the presence of locust bean gum was sequential, with mannanase activity preceding expression of alpha-galactosidase activity. The hemicellulase activities were purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, hydrophobic interaction chromatography, and ion-exchange and chromatofocusing techniques. The purified beta-D-mannanase is a dimeric enzyme (162 kilodaltons) composed of subunits having identical molecular weight (73,000). Maximal activity did not vary between pH 5.5 and 7.5. The beta-D-mannanase activity exhibited thermostability, retaining nearly full activity after incubation for 24 h at 70 degrees C and pH 6.5. The enzyme displayed high specificity for galactomannan substrates, with no-secondary xylanase or cellulase activity detected. Hydrolysis of locust bean gum yielded short oligosaccharides compatible with an endo mode of substrate depolymerization. Initial rate velocities of the mannanase activity displayed substrate inhibition and yielded estimates for Vmax and Km of 455 +/- 60 U/mg and 1.5 +/- 0.3 mg/ml, respectively, at 70 degrees C and pH 6.5. The alpha-galactosidase activity corresponded to a trimeric enzyme (247 kilodaltons) having subunits of identical molecular weight (82,000). The alpha-galactosidase had maximal activity at pH 7 to 7.5 and retained full activity after 24 h of incubation at 60 degrees C. The enzyme had only limited activity on galactomannan substrates as compared with hydrolysis of p-nitrophenyl alpha-D-galactose. Kinetics of p-nitrophenyl alpha-D-galactose hydrolysis yielded linear reciprocal plots corresponding to Vmax and Km of 195 +/- 10 U/mg and 0.25 +/- 0.02 mM, respectively, at 60 degrees C and pH 7. The characterization of the mannanase activity is consistent with its potential use in enzymatic bleaching of softwood pulps.

Drug Stability↗
PubMed1990

Extracellular beta-galactosidase activity of a Fibrobacter succinogenes S85 mutant able to catabolize lactose.

Fibrobacter succinogenes S85 is unable to grow with lactose as the source of carbohydrate, although it does exhibit low beta-galactosidase (EC 3.2.1.23) activity. Spontaneous mutants of strain S85 able to grow on lactose were isolated after spreading cells on a chemically defined agar medium with lactose as the carbohydrate source. A lactose-catabolizing isolate, designated L2, exhibited a sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profile and an immunoblot profile with polyclonal antibodies to whole cells of S85 which were identical to those observed for S85. Strain L2 exhibited both cell-associated and extracellular beta-galactosidase activity with either p-nitrophenyl-beta-D-galactopyranoside or lactose as the substrate. The cell-associated enzyme exhibited the greatest activity in the periplasmic space. Enzyme production was partially inhibited by glucose. The beta-galactosidase was activated by divalent cations and exhibited a pH optimum of 6.5. Analysis of the extracellular culture fluid revealed that glucose derived from the hydrolysis of lactose was used for growth, but galactose was not metabolized further. Cells were unable to take up the lactose analog, methyl-beta-D-thiogalactopyranoside. These data suggest that beta-galactosidase of F. succinogenes L2 cleaves lactose outside the cells and that the glucose released is catabolized while the galactose accumulates in the extracellular culture fluid.

Animals↗
PubMed1993

Conditions of formation, purification, and characterization of an alpha-galactosidase of Trichoderma reesei RUT C-30.

Trichoderma reesei RUT C-30 formed an extracellular alpha-galactosidase when it was grown in a batch culture containing lactose or locust bean gum as a carbon source. Short-chain alpha-galactosides (melibiose, raffinose, stachyose), as well as the monosaccharides galactose, dulcitol, arabinose, and arabitol, also induced alpha-galactosidase activity both when they were used as carbon sources (at a concentration of 1%) in batch cultures and in resting mycelia (at concentrations in the millimolar range). The addition of 50 mM glucose did not affect the induction of alpha-galactosidase formation by galactose. alpha-Galactosidase from T. reesei RUT C-30 was purified to homogeneity from culture fluids of galactose-induced mycelia. The active enzyme was a 50 +/- 3-kDa, nonglycosylated monomer which had an isoelectric point of 5.2. It was active against several alpha-galactosides (p-nitrophenyl-alpha-D-galactoside, melibiose, raffinose, and stachyose) and galactomannan (locust bean gum) and was inhibited by the product galactose. It released galactose from locust bean gum and exhibited synergism with T. reesei beta-mannanase. Its activity was optimal at pH 4, and it displayed broad pH stability (pH 4 to 8). Its temperature stability was moderate (60 min at 50 degrees C resulted in recovery of 70% of activity), and its highest level of activity occurred at 60 degrees C. Its action on galactomannan was increased by the presence of beta-mannanase.

Enzyme Induction↗
PubMed1999

Biochemical and phylogenetic analyses of a cold-active beta-galactosidase from the lactic acid bacterium Carnobacterium piscicola BA.

We are investigating glycosyl hydrolases from new psychrophilic isolates to examine the adaptations of enzymes to low temperatures. A beta-galactosidase from isolate BA, which we have classified as a strain of the lactic acid bacterium Carnobacterium piscicola, was capable of hydrolyzing the chromogen 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-Gal) at 4 degrees C and possessed higher activity in crude cell lysates at 25 than at 37 degrees C. Sequence analysis of a cloned DNA fragment encoding this activity revealed a gene cluster containing three glycosyl hydrolases with homology to an alpha-galactosidase and two beta-galactosidases. The larger of the two beta-galactosidase genes, bgaB, encoded the 76.8-kDa cold-active enzyme. This gene was homologous to family 42 glycosyl hydrolases, a group which contains several thermophilic enzymes but none from lactic acid bacteria. The bgaB gene from isolate BA was subcloned in Escherichia coli, and its enzyme, BgaB, was purified. The purified enzyme was highly unstable and required 10% glycerol to maintain activity. Its optimal temperature for activity was 30 degrees C, and it was inactivated at 40 degrees C in 10 min. The K(m) of freshly purified enzyme at 30 degrees C was 1.7 mM, and the V(max) was 450 micromol. min(-1). mg(-1) with o-nitrophenyl beta-D-galactopyranoside. This cold-active enzyme is interesting because it is homologous to a thermophilic enzyme from Bacillus stearothermophilus, and comparisons could provide information about structural features important for activity at low temperatures.

Amino Acid Sequence↗
PubMed2001

Purification and characterization of the recombinant Thermus sp. strain T2 alpha-galactosidase expressed in Escherichia coli.

The nucleotide sequence of the Thermus sp. strain T2 DNA coding for a thermostable alpha-galactosidase was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 474 amino acids (M(r), 53,514). The observed homology between the deduced amino acid sequences of the enzyme and alpha-galactosidase from Thermus brockianus was over 70%. Thermus sp. strain T2 alpha-galactosidase was expressed in its active form in Escherichia coli and purified. Native polyacrylamide gel electrophoresis and gel filtration chromatography data suggest that the enzyme is octameric. The enzyme was most active at 75 degrees C for p-nitrophenyl-alpha-D-galactopyranoside hydrolysis, and it retained 50% of its initial activity after 1 h of incubation at 70 degrees C. The enzyme was extremely stable over a broad range of pH (pH 6 to 13) after treatment at 40 degrees C for 1 h. The enzyme acted on the terminal alpha-galactosyl residue, not on the side chain residue, of the galactomanno-oligosaccharides as well as those of yeasts and Mortierella vinacea alpha-galactosidase I. The enzyme has only one Cys residue in the molecule. para-Chloromercuribenzoic acid completely inhibited the enzyme but did not affect the mutant enzyme which contained Ala instead of Cys, indicating that this Cys residue is not responsible for its catalytic function.

Amino Acid Sequence↗
PubMed1969

Survey of some Actinomycetales for alpha-galactosidase activity.

The enzyme alpha-galactosidase offers potential to (i) eliminate possibly the flatus-inducing factor(s) in edible beans, (ii) eliminate raffinose during beet-sugar processing, and (iii) determine raffinose analytically. Accordingly, 20 genera of the order Actinomycetales Buchanan 1917 were tested for evidence of alpha-galactosidase activity. Test filtrates were prepared with a medium containing D-galactose and soybean meal. Enzyme activity was demonstrated through cellulose thin-layer chromatography. Of 123 strains tested, 28 produced extracellular alpha-galactosidase. Almost all were streptomycetes. Members of the genera Actinoplanes Couch 1950, Micromonospora varphiOrskov 1923, and Promicromonospora Krasil'nikov et al. 1961 also exhibited alpha-galactosidase activity. Additional tests led to the selection of five strains whose filtrates degraded melibiose, raffinose, and stachyose but not lactose and sucrose. Tests also were made with several soybean preparations.

Actinomycetales↗
PubMed1977

Identification, separation, and preliminary characterization of invertase and beta-galactosidase in Actinomyces viscosus.

The initial step of disaccharide dissimilation by Actinomyces viscosus serotype 2 strain M-100 was studied. Sucrase activity was found in the 3,000 X g particulate fraction and the 37,000 X g soluble fraction of the cells, whereas lactase activity was found almost exclusively in the 37,000 X g soluble fraction. Neither sucrase nor lactase activity was appreciable in the culture liquor. Sucrose phosphorylase, alpha-glucosidase, and polysaccharide synthesis activities were not observed in the soluble cell fraction. The sucrase was identified as invertase (EC 3.2.1.26; beta-D-fructofuranoside fructohydrolase). The lactase was identified as beta-galactosidase (EC 3.2.1.23; beta-D-galactoside galactohydrolase). The enzymes in the 37,000 X g soluble fraction were separable by diethylamino-ethyl-cellulose chromatography, giving one beta-galactosidase peak and one major and one minor invertase peak. Acrylamide gel electrophoresis showed different electrophoretic mobilities of the enzymes. The molecular weight of the beta-galactosidase is about 4.2 X 10(5) and that of invertase is about 8.6 X 10(4). The beta-galactosidase has a Km for lactose of about 6 mM and a pH optimum between pH 6.0 and 6.5. The major invertase component has a Km for sucrose of about 71 mM and a pH optimum between pH 5.8 and 6.3.

Actinomyces↗
PubMed1970

Comparative study of isoenzyme formation of bacterial beta-galactosidase.

The enzyme beta-galactosidase was studied in crude extracts of Escherichia coli 3300, E. coli grown on a selenium and sulfur medium, Salmonella typhimurium F-lac, Serratia marcescens F-lac, S. marcescens P-lac, Proteus mirabilis F-lac, P. mirabilis P-lac, Aeromonas formicans, and Streptococcus lactis. The isoenzymes could be demonstrated by an alternative histochemical technique. Different isoenzyme patterns were found to be determined by the beta-galactosidase structural gene and not by the cytoplasm within which the beta-galactosidase was formed. In addition, the beta-galactosidases from strains which form isoenzymes were more stable to heat and urea treatments than the enzyme formed by those organisms which produce reduced amounts of, or no, isoenzyme.

Acrylates↗
PubMed1971

Properties of an extracellular -galactosidase secreted by Neurospora crassa.

An extracellular beta-galactosidase with an activity optimum at about pH 4 was found to occur in the filtrates of Neurospora crassa strain 74A when this mold was grown on certain sugars. This activity accounted for a substantial portion of the beta-galactosidase activity in the culture. The beta-galactosidase in the medium appeared to be a secreted, extracellular enzyme, not a product of cell lysis. The extracellular activity was found to have physical and kinetic properties similar to those of an intracellular beta-galactosidase previously found in Neurospora. Some conditions for the production and concentration of the enzyme are described.

Carbohydrate Metabolism↗
PubMed1971

Inducible and constitutive -galactosidase formation in cells recovering from protein synthesis inhibition.

Inducible and constitutive beta-galactosidase formation and radioactive amino acid incorporation were measured in cells recovering from various treatments which inhibit protein synthesis in the cell. Undelayed beta-galactosidase formation was found in stringent auxotrophs recovering from amino acid starvation, in cells recovering from glycerol or potassium starvation, and in bacteria recovering from puromycin treatment. Delayed beta-galactosidase formation was found in relaxed auxotrophs recovering from amino acid starvation and in prototrophs recovering from chloramphenicol or from tetracycline treatment. The length of this delay was directly proportional to the duration of the treatment. All cells recovering from the various treatments exhibited a slightly decreased rate of beta-galactosidase formation and an increase in radioactive amino acid incorporation.

Amino Acids↗
PubMed1975

Effect of glucose and its analogues on the accumulation and release of cyclic adenosine 3',5'-monophosphate in a membrane fraction of Escherichia coli: relation to beta-galactosidase synthesis.

Correlation between beta-galactosidase synthesis and cyclic adenosine 3',5'-monophosphate (cAMP) levels in a membrane fraction obtained from disrupted spheroplasts of Escherichia coli was investigated. Repression of beta-galactosidase synthesis in the membrane fraction by glucose-6-phosphate and by 2-deoxyglucose differed in sensitivity to reversal by cAMP. The difference between the two repressions could be due to the fact that glucose-6-phosphate inhibited severely the accumulation of exogenous [3-H]cAMP by the membrane fraction, whereas 2-deoxyglucose had little effect on the accumulation of the nucleotide. On the other hand, a quick decrease in the level of [3-H]cAMP preaccumulated in the membrane fraction resulted from addition of either glucose-6-phosphate or 2-deoxyglucose. Results reported here suggest that repression of beta-galactosidase synthesis is associated with anabrupt decrease in cAMP levels at the intramembranal sites where beta-galactosidase is synthesized, and the major, if not sole, mechanism which leads to instantaneous drop of cAMP level is via the release of cAMP, but not by degradation of the nucleotide since the membrane fraction retained less than 10 percent of cellular cyclic phosphodiesterase and the activity of the enzyme was not affected by repressing sugars.

Adenylyl Cyclases↗
PubMed1995

Analysis of a novel gene and beta-galactosidase isozyme from a psychrotrophic Arthrobacter isolate.

We have characterized a new psychrotrophic Arthrobacter isolate which produces beta-galactosidase isozymes. When DNA from this isolate was transformed into an Escherichia coli host, we obtained three different fragments, designated 12, 14, and 15, each encoding a different beta-galactosidase isozyme. The beta-galactosidase produced from fragment 12 was of special interest because the protein subunit was smaller (about 71 versus 116 kDa) than those typically encoded by the lacZ family. The isozyme encoded by fragment 12 was purified, and its activity and thermostability were examined. Although the enzyme is highly specific towards beta-D-galactoside substrates, its levels in the isolate do not increase in cells grown with lactose. Nucleotide sequence determination showed that the gene encoding isozyme 12 is not similar to the other members of the lacZ family but has regions similar to beta-galactosidase isozymes from Bacillus stearothermophilus and B. circulans. Addition of the isozyme 12 sequence to the database made it possible to examine these enzymes as possible members of a new, separate family. Our analysis of this new family showed some conserved amino acids corresponding to the lacZ acid-base catalytic region but no homology with the nucleophilic region. On the basis of these comparisons, we designated this a new lacG family.

Amino Acid Sequence↗
PubMed2003

Biochemical characterization of a beta-galactosidase with a low temperature optimum obtained from an Antarctic arthrobacter isolate.

A psychrophilic gram-positive isolate was obtained from Antarctic Dry Valley soil. It utilized lactose, had a rod-coccus cycle, and contained lysine as the diamino acid in its cell wall. Consistent with these physiological traits, the 16S ribosomal DNA sequence showed that it was phylogenetically related to other Arthrobacter species. A gene (bgaS) encoding a family 2 beta-galactosidase was cloned from this organism into an Escherichia coli host. Preliminary results showed that the enzyme was cold active (optimal activity at 18 degrees C and 50% activity remaining at 0 degrees C) and heat labile (inactivated within 10 min at 37 degrees C). To enable rapid purification, vectors were constructed adding histidine residues to the BgaS enzyme and its E. coli LacZ counterpart, which was purified for comparison. The His tag additions reduced the specific activities of both beta-galactosidases but did not alter the other characteristics of the enzymes. Kinetic studies using o-nitrophenyl-beta-D-galactopyranoside showed that BgaS with and without a His tag had greater catalytic activity at and below 20 degrees C than the comparable LacZ beta-galactosidases. The BgaS heat lability was investigated by ultracentrifugation, where the active enzyme was a homotetramer at 4 degrees C but dissociated into inactive monomers at 25 degrees C. Comparisons of family 2 beta-galactosidase amino acid compositions and modeling studies with the LacZ structure did not mimic suggested trends for conferring enzyme flexibility at low temperatures, consistent with the changes affecting thermal adaptation being localized and subtle. Mutation studies of the BgaS enzyme should aid our understanding of such specific, localized changes affecting enzyme thermal properties.

Antarctic Regions↗
PubMed1996

Uneven X inactivation in a female monozygotic twin pair with Fabry disease and discordant expression of a novel mutation in the alpha-galactosidase A gene.

We describe two female monozygotic (MZ) twins heterozygous for Fabry disease, an X linked disorder resulting from the deficient activity of alpha-galactosidase A. While one of the twins was clinically affected, the other was asymptomatic. Enzymatic assay of alpha-galactosidase in blood leucocytes, skin fibroblasts, Epstein-Barr virus transformed lymphoid cell lines, and hair follicles of the twins and their parents confirmed the heterozygous status of the twins and indicated that Fabry disease had occurred as a result of a de novo mutation. The son of the unaffected twin sister was shown to be hemizygous. Molecular analysis of the alpha-galactosidase A gene permitted the identification of an as yet undescribed point mutation at position 10182 of exon 5 which causes an Asp to Asn substitution at codon 231. Single strand conformation polymorphism (SSCP) analysis again showed the heterozygous status of the twins and a normal pattern in their parents. The basis for the discordant expression of this d novo mutation in the twins was investigated by studying their X inactivation status. Analysis of the inactive X specific methylation at the androgen receptor gene showed unbalanced inactivation in the twins' fibroblasts and in opposite directions. While the maternally derived X chromosome was preferentially active in the asymptomatic twin, the paternal X chromosome was active in the other, affected twin and was found in her hemizygotic nephew. These data suggest that the paternal X chromosome carries the de novo alpha-galactosidase A mutation and that uneven X inactivation is the underlying mechanism for disease expression in this novel female MZ twin pair. This is the first documented case of female twins discordant for Fabry disease.

Adult↗
PubMed2004

A study of the relationships of interactions between Asp-201, Na+ or K+, and galactosyl C6 hydroxyl and their effects on binding and reactivity of beta-galactosidase.

The interactions between Na+ (and K+) and Asp-201 of beta-galactosidase were studied. Analysis of the changes in Km and Vmax showed that the Kd for Na+ of wild type beta-galactosidase (0.36 +/- 0.09 mM) was about 10x lower than for K+ (3.9 +/- 0.6 mM). The difference is probably because of the size and other physical properties of the ions and the binding pocket. Decreases of Km as functions of Na+ and K+ for oNPG and pNPG and decreases of the Ki of both shallow and deep mode inhibitors were similar, whereas the Km and Ki of substrates and inhibitors without C6 hydroxyls remained constant. Thus, Na+ and K+ are important for binding galactosyl moieties via the C6 hydroxyl throughout catalysis. Na+ and K+ had lesser effects on the Vmax. The Vmax of pNPF and pNPA (substrates that lack a C6 hydroxyl) did not change upon addition of Na+ or K+, showing that the catalytic effects are also mediated via the C6 hydroxyl. Arrhenius plots indicated that Na+, but not K+, caused k3 (degalactosylation) to increase. Na+ also caused the k2 (galactosylation) with oNPG, but not with pNPG, to increase. In contrast, K+ caused the k2 values with both oNPG and pNPG to increase. Na+ and K+ mainly altered the entropies of activation of k2 and k3 with only small effects on the enthalpies of activation. This strongly suggests that only the positioning of the substrate, transition states, and covalent intermediate are altered by Na+ and K+. Further evidence that positioning is important was that substitution of Asp-201 with a Glu caused the Km and Ki values to increase significantly. In addition, the Kd values for Na+ or K+ were 5 to 8 fold higher. The negative charge of Asp-201 was shown to be vital for Na+ and K+ binding. Large amounts of Na+ or K+ had no effect on the very large Km and Ki values of D201N-beta-galactosidase and the Vmax values changed minimally and in a linear rather than hyperbolic way. D201F-beta-galactosidase, with a very bulky hydrophobic side chain in place of Asp, essentially obliterated all binding and catalysis.

Aspartic Acid↗