Kolmar, Gertrud (eigtl. G. K. Chodziesner)

Brandt, Marion

doi:10.1007/978-3-476-03702-2_185

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“…A+i + 0) and that the reaction The experiments demonstrate that binding of the inhibitor Dns-r,TrpOEt to rlchymotrypsin involves an additional slow step, which has so far not been observed in this system. As pointed out by Brandt et al [19]. the acylation of chymotrypsin with N-acetyl-L-tryptophan ethyl ester as substrate is pH-dependent and occurs in the time range from 10-100 SKI.…”

Section: Concentration Dependence Of' Relaxation Amplitudesmentioning

confidence: 94%

Two‐step Interaction of α‐Chymotrypsin with a Fluorescent Inhibitor

Rodier¹,

Ilgenfritz²

1982

European Journal of Biochemistry

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Bovine a-chyniotrypsin interacts with a dansylated inhibitor. I-dimethylaminonaphthalene-5-sulfonyl-D-tryptophan ethyl ester, to give a stable and strongly fluorescent derivative. The kinetics of the interaction of the enzyme with this inhibitor has been investigated by the temperature-jump method. It is shown that the complex formation proceeds in two steps. a binding step and a slow isomerisation. as deduced from a study of the concentration dependence of the relaxation times. The rate constants for the binding process are klz = 7.9 x 10' overall binding constant determined statically of 1740 M -' . leads to the determination of the kinetic parameters for the slow isomerisation k23 = 19 s-' and k32 = 9 s-'. Both values have the same order of magnitude a s the rate constants found for the pH-induced relaxation of some pancreatic proteins. This slow isomerisation can be related to internal m o t i y s of the active center of the enzyme.M-1 5 -1. and k-2, = 1 . 3 6~ 103 s-', giving a binding constant of 585 M-I. This last value. compared to thcThe interaction of proteins with ligands can cxhibit multistate kinetic behaviour. For example. the binding of a ligand to a protein. followed by a single isomerisation of the latter. will give, in general, two-step kinetic behaviour. These features were observed for the binding of proflavin to x-chymotrypsin [l]. the binding of tRNASer to seryl-tRNA synthetase [2]. or the binding of various ligands to 11-S acetylcholinesterase [3]. In many cases. as for example with pyruvate kinase [4], conformational changes can be observed without binding after a short perturbation of the equilibrium conditions.1 t seems well established that these conformational changes can occur in a broad time range from a few milliseconds for a simple isomerisation to some minutes for denaturation processes. For a given protein studied with different perturbation techniques with or without ligand, one can hope to establish a certain conformational change pattern.z-Chymotrypsin undergoes several transitions by interacting with substrate models or inhibitors or simply after a perturbation (pH or temperature) of the surrounding medium.Havsteen [ l ] has studied the interaction of a-chymotrypsin with proflavin. a competitive inhibitor. He reported twostep kinetic behaviour of the progress curve in temperaturejump experiments with isomerisation rate constants of about 500 s-' and 7000 s for the forward and back reaction rcspectively. Vandersijpen and Heremans [ 5 ] have reported high-pressure temperature-jump studies performed on the latter system and were unable to observe the two relaxation effects mentioned by Havsteen. More recently Hanai showed that a-chymotrypsin undergoes a one-step isomerisation after binding with benzeneboronic acid in the millisecond range [6].The folding of serine proteases trypsin, chymotrypsin and their related zymogens have been studied by various pertur- In the work reported here, we have found a slow isomerisation of r-chymotrypsin in the range of a tenth of a second. An ...

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Section: Concentration Dependence Of' Relaxation Amplitudesmentioning

confidence: 94%