The necessity of magnesium cation for acid assistance aglycone departure in catalysis by <i>Escherichia coli</i> (<i>lacZ</i>) β-galactosidase

Sinnott, Michael L.; Withers, Stephen G.

doi:10.1042/bj1750539

Cited by 45 publications

(11 citation statements)

References 34 publications

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“…The divalent cations Mg2" and Mn2" did not affect activity toward ONPG. This is in contrast to the observations that both the lacZ,f-galactosidase and the ebgA fl-galactosidase of E. coli require Mg2e for maximal activity (7,15). It is of some interest to note that if there were one active site per 68,000-molecular-weight subunit, the Kc,,t for BGase-III would be 1,140 s-1, very close to the value of 1,100 s-1 reported for hydrolysis of ONPG by lacZ,8-galactosidase of E. coli (14).…”

Section: J Bacteriolcontrasting

confidence: 71%

Properties of beta-galactosidase III: implications for entry of galactosides into Klebsiella

Hall¹

1980

J Bacteriol

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Klebsiella sp. strain CT-200 lacks both its plasmid-borne lac operon, which specifies ,-galactosidase I, and its chromosomal lac operon, which specifies flgalactosidase II, but it expresses a gene for a third,8-galactosidase, ,B-galactosidase Ill, constitutively. CT-200 was examined to determine whether there was a,galactoside permease associated with the ,B-galactosidase III gene. The failure of CT-200 to transport thiomethyl-fl-galactoside, o-nitrophenyl-fl-D-galactopyranoside, phenyl-,B-galactoside, lactulose, or galactosyl-arabinose was taken as evidence that B-galactoside permease is not part of a fi-galactosidase III operon. Optimal assay conditions for ,8-galactosidase III, whose activity was used as a measure of B-galactoside transport, are reported here, as are an improved purification method and some physical and catalytic properties of the enzyme not previously reported. ,-galactosidase III (BGase-III) of Klebsiella is a "cryptic" ,8-galactosidase synthesized by derivatives of strain RE1544. It was first detected in a lactose-negative mutant, strain RE1755, which had lost both of its lac operons and thus failed to synthesize ,8-galactosidases I and II (9, 13). BGase-III synthesis is induced by lactose (5, 9) and by methyl-,B-galactoside (5), but not by thiogalactosides, melibiose, or galactose (5, 9). Both the chromosomal and plasmid-borne lac operons include genes for permease, as well as for,B-galactosidase (13). Since both lactose and methyl-,f-galactoside are able to induce synthesis of BGase-III, it is clear that these sugars do enter the cell. It thus seemed reasonable to inquire whether there might be a permease gene associated with the gene for BGase-III. MATERIALS AND METHODS Bacterial strains. All strains were derived from Klebsiella ap. strain RE1544 (13). This strain is not designated specifically K. pneumonia or K. aerogenes because of the lack of agreement among different authorities as to what should be included in these taxonomic groups (9, 13). RE1755 is a Lac-Galmutant of RE1544 in which neither the chromosomal nor plasmid-borne lac operons produce active ,B-galactosidase (9, 13), but in which synthesis of BGase-III is induced by lactose and by methyl-,8-galactoside (5, 9). Strain CT-1 is a Lac+ Gal' spontaneous mutant of RE1755 in which synthesis of BGase-III is no longer inducible by lactose, although it is still inducible by methyl-,B-galactoside (5). Strain CT-200 is a mutant of strain CT-1 which synthesizes BGase-III constitutively. The isolation of strain CT-200 is described in the text. Strain CT-200 was the source of BGase-III for this study. Media. Minimal medium, consisting of phosphate

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Section: J Bacteriolcontrasting

confidence: 71%

Properties of beta-galactosidase III: implications for entry of galactosides into Klebsiella

Hall¹

1980

J Bacteriol

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“…The enzyme was unlikely to be saturated with substrate, since its extracellular concentration (0.7 M) was the same as the K m of ␤-galactosidase at low magnesium concentrations (18). Thus, higher concentrations of peptide can increase the rate of ONPG hydrolysis by increasing the extent to which the membrane is permeabilized.…”

Section: Discussionmentioning

confidence: 99%

Antibacterial and Antimembrane Activities of Cecropin A in Escherichia coli

Silvestro¹,

Weiser

Axelsen

2000

Antimicrob Agents Chemother

115

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The ability of cecropin A to permeabilize and depolarize the membranes of Escherichia coli ML-35p bacteria has been compared to its bactericidal activity in an extension of earlier studies performed on synthetic lipid vesicle membranes (L. Silvestro, K. Gupta, J. H. Weiser, and P. H. Axelsen, Biochemistry 36:11452-11460, 1997). Our results indicate that differences in the concentration dependences of membrane permeabilization and depolarization seen in synthetic vesicles are not manifested in whole bacteria. The concentration dependences of both phenomena roughly correlate with bactericidal activity, suggesting that the bactericidal mechanism of cecropin A is related to membrane permeabilization.Cecropin A is one of the most extensively studied antimicrobial polypeptides among the many that are produced by insects as components of their host defense systems against bacterial infection (6,7,14). While there is a broad consensus that the site of antibacterial action of these polypeptides is the plasma membrane, the precise mechanism-and the way in which they discriminate between bacterial and host cell membranes-remains unclear.Studies of cecropin A and related peptides have shown that cecropin A is a linear 37-residue polypeptide composed entirely of ordinary L-amino acids (21). It is unstructured in aqueous solution but has the potential to form ␣-helices in partially organic solvent (8,19). Early studies of their bactericidal mechanism suggested that cecropins bind to negatively charged membrane lipids and form a closely packed layer (20) or "carpet" of peptide (5, 15) which renders the membranes permeable. Later studies demonstrated that cecropins form partially selective ion channels (2). Evidence against a mechanism involving specific chiral receptors was provided by studies showing that analogues composed entirely of D-amino acids retained full activity (22). Models have been proposed for the ion channel formed by cecropins (3), and evidence is available indicating that they aggregate and assume a transbilayer orientation in membranes (12, 13). We have recently shown that the action of cecropin A on synthetic lipid vesicles is concentration dependent, forming ion channels at low peptide/lipid ratios and "pores" large enough to pass various probe molecules at higher peptide/lipid ratios (16). In that set of experiments we demonstrated that leakage of water-soluble probe molecules was "all or nothing," supporting the idea that the pores through which the probes escape are stable, water-filled transmembrane conduits.One limitation of many studies of antimicrobial peptide action is that they have been performed on synthetic lipid vesicles rather than bacteria. This is necessary in many cases to make particular types of studies feasible or interpretable. However, vesicle studies leave important questions open about the true nature of antimicrobial peptide action in a vastly more complex chemical milieu. Therefore, we have extended our earlier studies of cecropin A action on phospholipid vesicles to whole bacteri...

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“…It is of interest that early kinetic studies 40,75,76 led to the conclusion that a protein conformation change occurs after substrate binds. It was postulated that the acid catalytic group must move into position before it can interact with the galactosidic oxygen.…”

Section: Movement Of Substrate From the Shallow To The Deep Sitementioning

confidence: 99%

LacZ β‐galactosidase: Structure and function of an enzyme of historical and molecular biological importance

2012

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This review provides an overview of the structure, function, and catalytic mechanism of lacZ b-galactosidase. The protein played a central role in Jacob and Monod's development of the operon model for the regulation of gene expression. Determination of the crystal structure made it possible to understand why deletion of certain residues toward the amino-terminus not only caused the full enzyme tetramer to dissociate into dimers but also abolished activity. It was also possible to rationalize a-complementation, in which addition to the inactive dimers of peptides containing the ''missing'' N-terminal residues restored catalytic activity. The enzyme is well known to signal its presence by hydrolyzing X-gal to produce a blue product. That this reaction takes place in crystals of the protein confirms that the X-ray structure represents an active conformation. Individual tetramers of b-galactosidase have been measured to catalyze 38,500 6 900 reactions per minute. Extensive kinetic, biochemical, mutagenic, and crystallographic analyses have made it possible to develop a presumed mechanism of action. Substrate initially binds near the top of the active site but then moves deeper for reaction. The first catalytic step (called galactosylation) is a nucleophilic displacement by Glu537 to form a covalent bond with galactose. This is initiated by proton donation by Glu461. The second displacement (degalactosylation) by water or an acceptor is initiated by proton abstraction by Glu461. Both of these displacements occur via planar oxocarbenium ion-like transition states. The acceptor reaction with glucose is important for the formation of allolactose, the natural inducer of the lac operon.

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The necessity of magnesium cation for acid assistance aglycone departure in catalysis by Escherichia coli (lacZ) β-galactosidase

Cited by 45 publications

References 34 publications

Properties of beta-galactosidase III: implications for entry of galactosides into Klebsiella

Properties of beta-galactosidase III: implications for entry of galactosides into Klebsiella

Antibacterial and Antimembrane Activities of Cecropin A in Escherichia coli

LacZ β‐galactosidase: Structure and function of an enzyme of historical and molecular biological importance

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