Cryoenzymology of human plasmin catalysis: Comparison of cryosolvents and reactions with nitrophenyl ester and anilide substrates

Coll, R J; Fink, Anthony L.

doi:10.1016/0011-2240(87)90037-x

Cited by 5 publications

(4 citation statements)

References 22 publications

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“…Although many cosolvents are known as stabilizers, the cases of activation of the enzymatic reaction by them are rare. The increase in the rate constant of enzymatic catalysis caused by cosolvents can be explained by different mechanisms, for example, a shift of the dielectric constant of media in the presence of glycerol or sucrose [52], or participation of nucleophilic cosolvent as a reactant in a reaction of hydrolytic enzymes [53]. For reaction catalyzed by butyrylcholinesterase in solution with sucrose it was deduced that sucrose is excluded from the active site cavity and acts as a semi-permeable membrane at its entrance.…”

Section: Discussionmentioning

confidence: 99%

Mechanisms of Viscous Media Effects on Elementary Steps of Bacterial Bioluminescent Reaction

Lisitsa

Sukovatyi

Барцев

et al. 2021

IJMS

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Enzymes activity in a cell is determined by many factors, among which viscosity of the microenvironment plays a significant role. Various cosolvents can imitate intracellular conditions in vitro, allowing to reduce a combination of different regulatory effects. The aim of the study was to analyze the media viscosity effects on the rate constants of the separate stages of the bacterial bioluminescent reaction. Non-steady-state reaction kinetics in glycerol and sucrose solutions was measured by stopped-flow technique and analyzed with a mathematical model developed in accordance with the sequence of reaction stages. Molecular dynamics methods were applied to reveal the effects of cosolvents on luciferase structure. We observed both in glycerol and in sucrose media that the stages of luciferase binding with flavin and aldehyde, in contrast to oxygen, are diffusion-limited. Moreover, unlike glycerol, sucrose solutions enhanced the rate of an electronically excited intermediate formation. The MD simulations showed that, in comparison with sucrose, glycerol molecules could penetrate the active-site gorge, but sucrose solutions caused a conformational change of functionally important αGlu175 of luciferase. Therefore, both cosolvents induce diffusion limitation of substrates binding. However, in sucrose media, increasing enzyme catalytic constant neutralizes viscosity effects. The activating effect of sucrose can be attributed to its exclusion from the catalytic gorge of luciferase and promotion of the formation of the active site structure favorable for the catalysis.

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Section: Discussionmentioning

confidence: 99%

Mechanisms of Viscous Media Effects on Elementary Steps of Bacterial Bioluminescent Reaction

Lisitsa

Sukovatyi

Барцев

et al. 2021

IJMS

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“…Causes of defined breaks in an Arrhenius curve may include : (a) a temperature-induced conformation or 'phase' change in the enzyme; (b) a purely kinetic phenomenon, i.e., the replacement of one rate-limiting step by another at a critical temperature; (c) modification of the solvent structure at a critical temperature. The fact that the transitions observed for both kl and k2 occur near 0°C is not necessarily attributable to the nature of the solvent: in fact, studies of the reaction of human plasmin in a variety of mixed hydro-organic solvents [26] indicated that nonlinearity of the Arrhenius plot was due to a temperature-induced isomerization of the enzyme which was independent of the nature of the solvent. Considering, as a precedent, another electron transfer reaction, a 'break' between -15°C and -21 "C in the Arrhenius plot has been observed for the reaction of two-band cytochrome c?'…”

Section: Ejject Of Pressure On the Binding Of Co To Ferrous P460 Of Hmentioning

confidence: 99%

Effect of solvent, pressure and temperature on reaction rates of the multiheme hydroxylamine oxidoreductase

Balny

Hooper

1988

European Journal of Biochemistry

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Hydroxylamine oxidoreductase (HAO) of the ammonia-oxidizing bacterium Nitrosomonas catalyzes the oxidation: N H 2 0 H + H 2 0 + HNOz + 2e-+ 2 H'. The heme-like chromophore P460 is part of a site which binds substrate, extracts electrons and then passes them to the many c hemes of the enzyme. Reduction of the c hemes by hydroxylamine is biphasic with apparent first-order rate constants kl and k z . CO binds to ferrous P460 with apparent first-order rate constants, kl,co. In this work we have measured the binding of CO to ferrous P460 of hydroxylamine oxidoreductase and the reduction by substrate of some of the 24 c hemes of the ferric enzyme. These reactions have been studied in water and 40% ethylene glycol, at temperatures ranging from -15°C to 2 0 . 7 T and at hydrostatic pressures ranging over 0.1 -80 MPa. From the measurements, thermodynamic parameters A V* (activation volume), AG*, AH', and AS* have been calculated.CO binding. Binding of CO to ferrous P460 was similar to the binding of CO to ferrous horseradish peroxidase. The change of solvent had only a limited effect on A V* (-30 ml . mol-I), AG", AH* or AS* and did not cause an inflection in the Arrhenius plot or downward displacement of the linear relationship between In kl,co and P at a critical temperature. Binding was exothermic at high temperatures. The response of the binding of CO to solvent, temperature and pressure suggested that the CO binding site had little access to solvent and was not susceptible to change in protein conformation.Fast phase of reduelion of c hemes. Changing the solvent from water to 40% ethylene glycol resulted in a decrease from 90% to 50% in the relative number of c hemes reduced during the fast phase, an increase in activation volume from -3.6 ml . mol-' to 57 ml . mol-' and changes in other thermodynamic parameters. The activation volume increased with decreasing temperature. The Arrhenius plot had a downward inflection at about 0°C and, in water or ethylene glycol, the linear dependence of In kl on P was displaced downwards as the temperature changed from 3.5"C to -15°C.Slow phase of reduction of c hemes. Changing the solvent from water to 40% ethylene glycol resulted in an increase in the relative number of c hemes reduced during the slow phase from 10% to 50%. The activation volume, which was not measurable in water because of the low absorbance change, was -30 ml . mo1-l in ethylene glycol. The activation volume increased with increasing temperature. The Arrhenius plot had a downward inflection at about 0 "C yet the linear dependence of In k2 on P had no downward displacement in this temperature range. At high pressure and low temperatures the rate of heme reduction increased with decreasing temperature. In contrast to the reaction of CO with the substrate site, the results suggested that changes in enzyme conformation imposed by changes in solvent, temperature or pressure affected the rates of intramolecular electron transfer from the substrate site to c hemes.Hydroxylamine oxidoreductase from the ammonia-oxidizin...

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“…The increase in k, with increasing methanol concentration implies that the rate-limiting step is the breakdown of the intermediate I in Scheme I. As discussed in detail elsewhere (Coll & Fink, 1987), the expression for K, includes terms from the microscopic rate constants and is given by which reduces to Consequently, there is an additional component to the increase in K, with increasing methanol concentration that comes from the term in the numerator and thus parallels the observed increase in k,,,.…”

Section: Discussionmentioning

confidence: 84%

“…The increase in kat with increasing methanol concentration implies that the rate-limiting step is the breakdown of the intermediate 1 in Scheme I. As discussed in detail elsewhere (Coll & Fink, 1987), the expression for Km includes terms from the microscopic rate constants and is given by…”

Section: Discussionmentioning

confidence: 99%

Ribonuclease structure and catalysis: effects of crystalline enzyme, alcohol cryosolvents, low temperatures, and product inhibition

Fink¹,

Kar²,

Kotin³

1987

Biochemistry

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In order to determine the necessary conditions to stabilize intermediates in ribonuclease A catalysis at subzero temperatures for structural studies, we have examined the suitability of alcohol-based cryosolvents. On the basis of thermal denaturation transition curves, the enzyme is in the native conformation in high concentrations of ethanol and methanol, provided the temperature is suitably low. The effects of methanol on the catalytic properties for the hydrolysis for mono- and dinucleotide substrates also are consistent with the absence of adverse effects of the cosolvent. Significant methanolysis occurs in the presence of methanol as cosolvent. The kinetics of 2',3'-CMP hydrolysis are complicated by severe competitive product inhibition, both in aqueous and in methanolic solvents, accounting for the previously observed effect of substrate concentration on the observed Km. Computer-aided analysis allowed the determination of the inhibition constant as a function of experimental parameters. The reaction of ribonuclease A with 2',3'-CMP was investigated at subzero temperatures. The turnover reaction could be made negligible at temperatures below -60 degrees C at pH 3-6 in 70% methanol and below -35 degrees C at pH 2.1. The rate of the catalytic reaction with crystalline enzyme was compared to that of enzyme in solution for both 2',3'-CMP and the dinucleotide CpC. The rates were 50- and 200-fold slower, respectively, in the crystal. These investigations allowed calculation of the necessary conditions for NMR and X-ray diffraction experiments on the trapped enzyme--substrate intermediate.

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Cryoenzymology of human plasmin catalysis: Comparison of cryosolvents and reactions with nitrophenyl ester and anilide substrates

Cited by 5 publications

References 22 publications

Mechanisms of Viscous Media Effects on Elementary Steps of Bacterial Bioluminescent Reaction

Mechanisms of Viscous Media Effects on Elementary Steps of Bacterial Bioluminescent Reaction

Effect of solvent, pressure and temperature on reaction rates of the multiheme hydroxylamine oxidoreductase

Ribonuclease structure and catalysis: effects of crystalline enzyme, alcohol cryosolvents, low temperatures, and product inhibition

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