Application of secondary .alpha.-deuterium kinetic isotope effects to studies of enzyme catalysis. Glycoside hydrolysis by lysozyme and .beta.-glucosidase

Dahlquist, Frederick W.; Rand-Meir, Tsafrira; Raftery, Michael A.

doi:10.1021/bi00838a045

Cited by 106 publications

(54 citation statements)

References 21 publications

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“…Lysoryme I n an early application of secondary isotope effects to the study of enzyme reactions, Dahlquist et al showed that the lysozyme-catalyzed hydrolysis of a phenyl-8-D-glucopyranoside, containing deuterium a t C-1, is characterized by an isotope effect of 1.11. This isotope effect compares to values of 1.13 and 1.03 observed for acid (SN-1) and base (SN-2) hydrolysis (165). The similarity of isotope effects observed for the lysozyme-and acid-catalyzed reactions led Dahlquist et al to conclude considerable carbonium character at the transition state in the reaction catalyzed by lysozyme.…”

Section: B Glycosidasesmentioning

confidence: 71%

Kinetic Isotope Effects in Enzymology

Klinman

1978

Advances in Enzymology - And Related Areas of Molecular Biology

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Section: B Glycosidasesmentioning

confidence: 71%

Kinetic Isotope Effects in Enzymology

Klinman

1978

Advances in Enzymology - And Related Areas of Molecular Biology

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“…Phillips and coworkers (17,18) have proposed a charged species, a carbonium ion, for the activated complex of lysozyme. Also, Dahlquist, Rand-Mier, and Raftery (19) have found that the lysozyme reaction has carbonium ion character. The small value found for AV* is at least consistant with the supposition that the enzyme makes no major changes in conformation when it combines with the substrate to form the activated complex.…”

Section: Resultsmentioning

confidence: 99%

Hydrostatic Pressure and Ionic Strength Effects on the Kinetics of Lysozyme

Neville

Eyring

1972

Proc. Natl. Acad. Sci. U.S.A.

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The kinetics of the reaction catalyzed by egg-white lysozyme from hen eggs (EC 3.2.1.17) was investigated by measurement of the decrease in turbidity of suspensions of dried Micrococcus luteus cells. The substrate and NaCi concentration were varied, as well as the hydrostatic pressure (1-476 atm). A plot of the initial velocity against the reciprocal of the substrate concentration gave straight lines. From these plots values for the maximum velocity, V, and the apparent Michaelis constant, Kim as well as the effect of NaCl concentration were determined. The maximum velocity showed a pronounced maximum when plotted over a range of NaCl concentration from 0-0.208 M. The volume change ofactivation, A V*, calculated from V, was negative, and decreased in absolute value from -8 to about 0 cm'/mol as the NaCI molarity was increased over the above range. This effect is explained as due to a different number of salt molecules being built into activated complexes with each of these activated complexes reacting at its own characteristic rate. Strong pressure-dependence of enzyme catalysis indicates appreciable conformational changes of the enzyme as it is incorporated into the activated complex. The volume change causing the pressure effect arises principally from changes in the number of ionized species with the accompanying electrostriction.The influence of hydrostatic pressure on enzymes and cellular reactions has been extensively investigated, and the theoretical basis for interpretation of the effect is well known (1-4). This report is an investigation of the effect of changes in ionic strength and hydrostatic pressure on lysozyme kinetics. EXPERIMENTAL PROCEDURETwice-crystallized, salt-free egg-white lysozyme (N-acetylmuramide glycanohydrolase, EC 3.2.1.17), lot number LYSF8DB, was obtained from Worthington Biochemical Corp., Freehold, N.J. The purity of the enzyme was determined by ion-exchange chromatography on amberlite CG-50, according to the method of Tallan and Stein (5), and on BioRex 70, according to the method of Stevens and Bergstrom (6); the enzyme was more than 95% pure, and was used without further purification.The substrate used was dried Micrococcus luteus(leisodeikticus) cells, lot numbers T-2594 and U-1933, obtained from Mann Research Labs., New York, N.Y.For the enzyme assay at 1-and 476-atm hydrostatic pressure, the method of Shugar (7) was used, as modified (8). A Cary 14 spectrophotometer was used to record the reaction.Initial slopes were obtained by drawing tangent lines to the tracings, and from these slopes initial velocities were obtained. Each initial velocity was measured in quadruplicate. One * To whom reprint requests should be sent. enzyme unit is defined as the change of absorbancy equal to 0.001 at a wavelength of 450 nm.The pressure experiments were done in a medium-pressure optical absorption cell, model number 41-11545, made by the American Instrument Co., Silver Spring, Md. The change of the log of the rate with respect to pressure was linear over the range of pressure st...

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“…Model building further suggested that it would be difficult to fit the monosaccharide unit occupying the D position in its most stable chair conformation into the protein superstructure without distorting the ring toward the half chair favored by the sp2 transition state. Thus, the enzyme was postulated to act in part by means of a substrate distortion mechanism, whereby part of the substrate intrinsic binding energy is by secondary hydrogen isotope effects (32,33) and by oxygen-18 leaving group kinetic isotope effects (34). The latter group concluded that the transition state for the reaction resembled a nearly fully formed oxocarbocation.…”

Section: Discussionmentioning

confidence: 99%

Site-directed mutagenesis of the catalytic residues Asp-52 and Glu-35 of chicken egg white lysozyme.

Malcolm

Rosenberg

Corey

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

172

129

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The roles of the catalytic active-site residues aspartic acid-52 and glutamic acid-35 of chicken lysozyme (EC 3.2.1.17) have been investigated by separate in vitro mutagenesis of each residue to its corresponding amide (denoted as D52N and E35Q, respectively). The mutant enzyme D52N exhibits =5% of the wild-type lytic activity against Micrococcus luteus cell walls, while there is no measurable activity associated with E35Q (0.1% ± 0.1%). The measured dissociation constants for the chitotriose-enzyme complexes are 4.1 ,LM (D52N) and 13.4 pzM (E35Q) vs. 8.6 ,uM for wild type, indicating that the alterations in catalytic properties may be due in part to binding effects as well as to direct catalytic participation of these residues. The mutant lysozymes have been expressed in and secreted from yeast and obtained at a level of =a5 mg per liter of culture by high-salt elution from the cell walls.Chicken egg white lysozyme (CEWL; EC 3.2.1.17) has a distinguished history in the field of mechanistic enzymology. It was the first enzyme for which an atomic resolution x-ray structure was published (1), and the essence of the presently accepted catalytic mechanism was proposed almost exclusively from the structural information (2). The enzyme has additionally served as an important paradigm for studies in (i) the physical biochemistry of polydentate liganding, whereby the individual contributions to the several binding subsites have been separately evaluated (3); (ii) molecular evolution investigations (4, 5); and (iii) immunochemical studies (6). The enzyme is thus an attractive candidate for modification by site-directed mutagenesis, because the availability of specifically mutated constructs will have the potential to contribute significantly to all of the above investigations, as well as to studies on protein folding and stability. We report here the relatively high-efficiency expression of Strains. Plasmids were propagated in HB101 grown in LB medium (7). M13mpl8 subclones were grown in JM103 and in RZ1032 for mutagenesis (8). Saccharomyces cerevisiae strain GRF180 is leu2-3,-J12, ura3-52, his4-580, can (cir") and was derived from GRF18 by curing this strain of its endogenous 2-,um circle (9, 10). Escherichia coli and S. cerevisiae transformations were carried out as described (11,12).Plasmids. Plasmid pAB24 is a yeast-E. coli shuttle vector derived from pBR322 and pJDB219 (13). It contains the complete 2-gm circle and the LEU2-D and URA3 genes for selection and replication in yeast and pBR322 sequences and sequences for selection and replication in E. coli (14). Plasmid pAGAP1 is a derivative of pPGAP1 (15) that has the GAPDH-491 promoter replaced with a hybrid ADH2-GAPDH promoter fusion (14). Plasmid pLS1023 containing a CEWL cDNA clone (16) was the generous gift of G. Schutz (University of Cologne). DNA sequencing revealed two discrepancies between pLS1023 and the published sequence, which were repaired by site-directed mutagenesis.Site-Directed Mutagenesis of the Active Site. Two 21-base synthetic primers ...

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Application of secondary .alpha.-deuterium kinetic isotope effects to studies of enzyme catalysis. Glycoside hydrolysis by lysozyme and .beta.-glucosidase

Cited by 106 publications

References 21 publications

Kinetic Isotope Effects in Enzymology

Kinetic Isotope Effects in Enzymology

Hydrostatic Pressure and Ionic Strength Effects on the Kinetics of Lysozyme

Site-directed mutagenesis of the catalytic residues Asp-52 and Glu-35 of chicken egg white lysozyme.

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