Structure and activity of trypsin in reverse micelles

Self Cite

Reverse micelles, formed in isooctane/alcohol by phosphatidylcholines of variable chain length (i.e. 6, 7 or 8 C atoms in the fatty acid moiety) have been studied, mostly in relation to their capability of solubilizing trypsin and a-chymotrypsin. It has been found that the capability of the lecithin reverse micellar systems to solubilize water is strongly affected by the chain length of the alkyl group and by the alcohol used as co-surfactant. The C8-lecithin system, i.e. 1,2-dioctanoyl-sn-glycero-3-phosphocholine, in isooctane/hexanol is the system which affords the maximal solubilization of water (up to w, 60, where w, = [H,O]/[lecithin]) and of the enzymes. The water of the water pool of lecithin reverse micelles has been investigated by 'H-NMR; the proton chemical shift as a function of w, was found to be similar to the case of reverse micelles formed by the well known negatively charged surfactant sodium bis(2-ethylhexyl sulfosuccinate). 'P-NMR studies show that the ionization behavior of phosphate groups is similar to that in bulk water, suggesting no anomaly in the pH behavior of this water pool. The stability of trypsin and a-chymotrypsin in the various lecithin reverse micellar system is similar and occasionally better than that in aqueous solution. The same holds for the kinetic behavior (k,,, and K, have been determined for a few systems). The bell-shaped curve of the pH/activity profile in lecithin reverse micelles is, for both enzymes, shifted towards more alkaline values with respect to water. Bell-shaped curves are also obtained when studying the influence of w, on the enzyme activity, with an optimal w, which is in the range 7 -10, a surprisingly small value considering that we are dealing with hydrolases. Circular dichroic studies have been carried out in order to correlate the activity with the protein conformation: for both enzymes, generally no marked perturbations appear as a consequence of the solubilization in the lecithin reverse micelles, but conditions can be found under which significant alterations are present. Certain properties of the two enzymes, which in water solution are very similar, become sharply different in reverse micelles, showing that occasionally the micellization is able to enhance the relatively small structural differences between the two proteins.Enzymes in reverse micelles have enjoyed much interest over the last few years [I -41. One of the arguments generally advanced in favor of the relevance of this work for enzymology at large is that the microenvironment of proteins in reverse micelles simulates the microenvironment of proteins in biological membranes. The validity of the above analogy would obviously be stronger if the surfactant used for the reverse micelles were a biological surfactant. Generally however the surfactants used for micellar enzymology are sodium bis(2-ethylhexylsulfosuccinate) (AOT) or cetyltrimethylammonium bromide (CeMe,NBr) and only a few others [l -41. Reverse micelles formed by phospholipids have not as yet received so much attention, ...

Section: Kinetic Studiessupporting

confidence: 74%

Section: The P H Dependence Of the Enzymatic Activitymentioning

confidence: 98%

Section: The P H Dependence Of the Enzymatic Activitymentioning

confidence: 99%

See 1 more Smart Citation

The behavior of proteases in lecithin reverse micelles

Peng

Luisi

1990

Self Cite

“…IA). The same has been found for trypsin [4]. This observation is a direct consequence of the drastic changes in k2 and K, observed upon solubilizing the enzyme in reverse micelles (see below).…”

Section: Results and Disussionsupporting

confidence: 52%

Kinetic behaviour of α‐chymotrypsin in reverse micelles

Mao

Walde

Luisi

1992

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At the aim of investigating whether the early rapid phase of enzyme turnover is different in reverse micelles compared with bulk water, the kinetic properties of a-chymotrypsin have been studied in reverse micelles formed by sodium bis(2-ethylhexyl)sulfosuccinate in isooctane. Pre-steady state and steady-state kinetic constants, in water and in reverse micelles, have been determined by stoppedflow spectrophotometry for the hydrolysis of two substrates, namely acetyl-l-tryptophan-pnitrophenyl ester andp-nitrophenyl acetate. It has been shown that, for both substrates, the acylation rate constant (k,) is very much lower in reverse micelles than in water. However, the deacylation rate constant (k3) and the turnover number (kca,) are not significantly changed in reverse micelles with respect to bulk water. Therefore, despite considerable rate changes in the acylation step, deacylation is rate limiting both in water as well as in reverse micelles, under the experimental conditions used.The present paper reports a first investigation of the individual rate constants in the kinetics of cr-chymotrypsin in reverse micelles using stopped-flow spectrophotometry. Surprisingly, despite the large number of papers devoted to micellar enzymology (for recent reviews see, for example, [l -3]), stopped-flow spectrophotometry has, to date, not been used for enzymes in reverse micelles, with the cxception of one earlier attempt [4]. The study of the early phase of the enzyme cycle can provide information on the enzyme mechanism, and in particular be used to clarify the nature of the rate-limiting step associated with the enzyme turnover. Some enzyme/substrate systems in aqueous solution show, for example, a 'burstphase' [5] prior to the steady-state zero-order time progress. a-Chymotrypsin-catalyzed hydrolysis of esters is one such case, reflecting the fact that the rate-limiting step is at the level of the deacylation step [5].It seemed, therefore, particularly interesting to investigate the stopped-flow kinetics in reverse micelles, to determine whether the localization of the enzyme in a reverse micelle would change this mechanistic situation.The reverse-micellar system used was sodium (2-ethylhexy1)-sulfosuccinate (AOT) in isooctane; we have focussed our study on two p-nitrophenyl ester substrates, namely acetyl-L-tryptophan-p-nitrophenyl ester (Ac-Trp-ONp) and p-nitrophenyl acetate (Ac-ONp). In both cases, the a-chymo- trypsin-mediated substrate hydrolysis in aqueous systems proceeds through the following scheme [S] :where E is the enzyme, S the p-nitrophenyl ester substrate, ES the Michaelis complex, EAc the acylcnzyme and PI, Pz the two products (p-nitrophenol and acetyl-L-tryptophan or acetic acid). For both ester substrates, deacylation is the ratelimiting step in aqueous solution under neutral or slightly alkaline conditions [6, 71; in other words k2 % kJ. MATERIALS AND METHODS ReagentsAOT (approximately 99% pure) was from Sigma. Its purity was checked by recording the ultraviolet absorption spectrum in isooctane and by t...

“…The variation of their radii from 0.5-13 nm is directly related to the waterhrfactant molecular ratio W,, (Bru et al, 1995). Many encapsulations of enzymes (achymotrypsin, lysozyme, myoglobin) in bis-(2-ethylhexyl) sodium sulfosuccinate surfactant have been reported (Walde et al, 1988 ;Perez-Gilabert et al, 1992). The strength of interactions between enzyme and surfactant can be modulated by the modification of the W,, ratio.…”

Section: Circular Dichroismmentioning

confidence: 99%

Conformational and Associative Behaviours of the Third Helix of Antennapedia Homeodomain in Membrane‐Mimetic Environments

Berlose

Convert

Derossi

et al. 1996

160

134

The third helix of antennapedia homeodomain pAntp-(43 -58) can translocate through cell membrane and has been used as an intracellular vehicle for delivering peptides and oligonucleotides. The conformational and associative behaviour of two peptidic vectors pAntp-(43 -58) and [Pro50]pAntp-(43 -58) has been analyzed by different biophysical methods. pAntp-(43 -58) adopts an amphipathic helical structure in 30 % (by vol.) hexafluoroisopropanol, in perfluoro-tert-butanol and in the presence of SDS micelles.CD spectra indicate that the conformation of [Pro50]pAntp-(43 -58) in contrast to pAntp-(43 -58) is independent of the media used. 'H-NMR spectroscopy in SDS micelles or in perfluoro-tert-butanol allows detection of aggregated peptides probably in a ribbon 2, type conformation. These conformations became the predominant structure when Gln50 was replaced by ProSO. Interproton-distance restraints derived from NOE measurements have been classified in two groups corresponding to two types of structures: a-helix and essentially extended structures. Consecutive CHa(i)lCHa(i+ 1) NOES are only compatible with aggregates. Simulated annealing calculation of dimeric structure agrees with 4 and y/ angles in the p-sheet and y-turn regions. Fluorescence spectroscopy analysis has shown that the indole groups of both peptides penetrate into SDS micelles; both peptides also induce the formation of micelles at very low concentration of SDS (20 pM). Similar interaction was observed with reverse-phase micelles made of bis(2-ethyhexyl) sodium sulfosuccinate and small unilamellar vesicles (SUV) made of a mixture of phosphatidylcholine/phosphatidylserine. 3'P-NMR of vesicles (SUV and large unilamellar vesicles) indicated that the addition of pAntp analogues did not affect the size of phosphatidylcholine/phosphatidylserine vesicles. The addition of pAntp analogues to lipidic dispersions modulates lipid polymorphism in different ways depending on the mixtures of acidic lipids.