The Kinetics of the α-Chymotrypsin-Catalyzed Hydrolysis of p-Nitrophenyl Acetate<sup>*</sup>

Kézdy, Ferenc J.; Bender, Myron L.

doi:10.1021/bi00912a021

Cited by 265 publications

(127 citation statements)

References 27 publications

(25 reference statements)

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“…The kinetic constants obtained in the present study are summarized in Tables 1 and 2. First, the values which we determined for Ac-Trp-ONp and Ac-ONp in solution (pH 8) are in fair agreement with the values reported in the literature under comparable conditions [7,15,161. Although for both substrates deacylation is the rate-limiting step, (k, $ k 3 -.…”

Section: Methodssupporting

confidence: 88%

Kinetic behaviour of α‐chymotrypsin in reverse micelles

Mao

Walde

Luisi

1992

European Journal of Biochemistry

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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...

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Section: Methodssupporting

confidence: 88%

Kinetic behaviour of α‐chymotrypsin in reverse micelles

Mao

Walde

Luisi

1992

European Journal of Biochemistry

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“…43,44 We envisage improving the efficiency and selectivity of our designs in the future through directed evolution of residues close to the active site, 12 and/or via the precise installation of a surrogate for the oxyanion hole to stabilize the tetrahedral intermediates. 19 In conclusion, this is the first report of catalytically active Cys-His-Glu triads being installed into a completely de novo protein framework.…”

Section: Maldi-tof Ms At [M+84] Da (Simentioning

confidence: 99%

Installing hydrolytic activity into a completely de novo protein framework

et al. 2016

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Designing enzyme-like catalysts tests our understanding of sequence-tostructure/function relationships in proteins. Here, we install hydrolytic activity predictably into a completely de novo and thermo-stable -helical barrel, which comprises 7 helices arranged around an accessible channel. We show that the lumen of the barrel accepts 21 mutations to functional polar residues. The resulting variant, which has cysteine-histidine-glutamic acid triads on each helix, hydrolyses pnitrophenyl acetate with catalytic efficiencies matching the most-efficient redesigned hydrolases based on natural protein scaffolds. This is the first report of a functional catalytic triad engineered into a de novo protein framework. The flexibility of our system also allows the facile incorporation of unnatural side chains to improve activity and probe the catalytic mechanism. Such predictable and robust construction of truly de novo biocatalysts holds promise for applications in chemical and biochemical synthesis.(134 words)

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“…c An absorbance coefficient of 5.12 mM cm Ϫ1 at 400 nm for p-nitrophenol at pH 6.5 was calculated from the published pK a and absorbance coefficient of the p-nitrophenolate anion (22).…”

Section: Resultsmentioning

confidence: 99%

Leishmania major Elongation Factor 1B Complex Has Trypanothione S-Transferase and Peroxidase Activity

Vickers

Wyllie

Fairlamb

2004

Journal of Biological Chemistry

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In the Trypanosomatidae, trypanothione has subsumed many of the roles of glutathione in defense against chemical and oxidant stress. Crithidia fasciculata lacks glutathione S-transferase, but contains an unusual trypanothione S-transferase activity that is associated with eukaryotic translation elongation factor 1B (eEF1B). Here we describe the cloning, expression, and reconstitution of the purified ␣, ␤, and ␥ subunits of eEF1B from Leishmania major. Individual subunits lacked trypanothione S-transferase activity. Only eEF1B, formed by reconstitution or co-expression of the three subunits, was able to conjugate a variety of electrophilic substrates to trypanothione or glutathionylspermidine, but not glutathione. In contrast to the C. fasciculata eEF1B, the L. major enzyme also displayed peroxidase activity against a variety of organic hydroperoxides. The enzyme showed no activity with hydrogen peroxide and greatest activity with linoleic acid hydroperoxide (1 unit mg ؊1 ). Kinetic studies suggest a ternary complex mechanism, with K m values of 140 M for trypanothione and 7.4 mM for cumene hydroperoxide and k cat ‫؍‬ 25 s ؊1 . Immunofluorescence studies indicate that the enzyme may be localized to the surface of the endoplasmic reticulum. These results suggest that, in addition to its role in protein synthesis, the Leishmania eEF1B may help protect the parasite from lipid peroxidation.Parasitic protozoa of the family trypanosomatidae cause disease and death throughout the tropical and subtropical world. Trypanosoma brucei infections are estimated to cause ϳ400,000 cases of sleeping sickness per year; Trypanosoma cruzi, the cause of Chagas' disease, chronically infects ϳ17 million people and Leishmania spp. are thought to cause 2 million cases of leishmaniasis per year. 1 Current chemotherapies for these diseases are, on the whole, ineffective and toxic (2, 3), whereas effective vaccines may never be developed. The development of effective treatments for these infections is therefore an urgent necessity.New anti-parasitic drugs can be developed from inhibitors of biochemical pathways that are essential for parasite survival but absent from the host. One such target is the thiol metabolism of trypanosomatids. Uniquely, this is dependent upon trypanothione (N 1 ,N 8 -bis(glutathionyl)spermidine or T[SH] 2 ) 2 (4), whereas their human hosts use glutathione ((␥-L-glutamyl-L-cysteinylglycine or GSH). Trypanothione is involved in protective processes, defending against oxidative stress through peroxidase systems (5), against reactive aldehydes through the glyoxalase system (6, 7), and against toxic xenobiotics through the trypanothione S-transferase (TST) (8). All these processes depend upon trypanothione being maintained in its dithiol form by trypanothione reductase. This NADPH-dependent flavoenzyme is essential to the trypanosomatids (9, 10) as, in these organisms, it is the only known route for the transfer of reducing equivalents from NADPH to low molecular mass thiols. In addition, because these parasites lack an...

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The Kinetics of the α-Chymotrypsin-Catalyzed Hydrolysis of p-Nitrophenyl Acetate^*

Cited by 265 publications

References 27 publications

Kinetic behaviour of α‐chymotrypsin in reverse micelles

Kinetic behaviour of α‐chymotrypsin in reverse micelles

Installing hydrolytic activity into a completely de novo protein framework

Leishmania major Elongation Factor 1B Complex Has Trypanothione S-Transferase and Peroxidase Activity

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The Kinetics of the α-Chymotrypsin-Catalyzed Hydrolysis of p-Nitrophenyl Acetate*

Cited by 265 publications

References 27 publications

Kinetic behaviour of α‐chymotrypsin in reverse micelles

Kinetic behaviour of α‐chymotrypsin in reverse micelles

Installing hydrolytic activity into a completely de novo protein framework

Leishmania major Elongation Factor 1B Complex Has Trypanothione S-Transferase and Peroxidase Activity

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The Kinetics of the α-Chymotrypsin-Catalyzed Hydrolysis of p-Nitrophenyl Acetate^*