Hydrostatic Pressure and Ionic Strength Effects on the Kinetics of Lysozyme

Abstract

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...

The Lysozyme Inhibitor Thionine Acetate Is Also an Inhibitor of the Soluble Lytic Transglycosylase Slt35 from Escherichia coli

“…Due to the high concentration of the enzyme used here, lysozyme did not follow Michaelis–Menten kinetics, so the data were instead fit to the Morrison equation for tight binding of the substrate [ 41 ], giving an apparent K m of 0.034 mg mL −1 cell suspension. This is in keeping with previous studies, which found K m values in the region of 0.01–0.05 mg mL −1 for M. lysodeikticus cell suspension [ 42 ], and a K d value of 14 μM for chitotriose [ 43 ]. As Slt35 displayed both substrate inhibition and tight substrate binding, we were unable to obtain good fits for the experimental data.…”

Temperature-dependent effects of high pressure on the bioluminescence of firefly luciferase

“…The maximal light output was observed at about 22.5°C. (Neville and Eyring, 1972). At the present experimental condition where the system is in a quasi-equilibrium state, we assume that the overall reaction leading to light emission proceeds as if governed by a single specific-reaction rate constant k'.…”

“…The rate process measures the concentration of activated complex because the complexes always decompose at the same rate, kT/h (Neville and Eyring, 1972). The activated complex is in equilibrium with its environment and is composed of a species whose heat content is changing with temperature.…”

“…The pressure effect must be evaluated at a steady state (Neville and Eyring, 1972). The kinetics of initial burst of light and the secondary low luminescence is complex and the assignment of steady state is unclear.…”

Parameter Reliability and Understanding Enzyme Function

“…Sensor methods have been developed [ 81 ] and used to monitor ionic-strength changes in human kidney cells [ 82 ], but the possible effects of ionic strength on metabolism have been often ignored in kinetic and simulation studies. Although there have been some studies that indicate the effects of ionic strength on specific enzymes [ 83 , 84 , 85 ] and how it may affect intracellular pressure, protein folding, and aggregation [ 86 , 87 ], it has proved difficult to disentangle ionic-strength effects from those of the specific ionic species involved [ 26 , 86 ]. Ideally, any assay medium used for in vitro studies should mimic the cellular ionic strength, but that aspect has often been ignored.…”