Unfolding kinetics of bovine trypsinogen.


Abstract

The unfolding kinetics of bovine trypsinogen were studied by a fluorescence-detected stopped-flow technique at pH 5.8. Trypsinogen unfolding appeared to be a rather complex reaction. Two phases, fast (with a time constant in the millisecond range) and slow, were detected in the range 2-7 M guanidium chloride (GdmCl). The natural logarithm of the rate constant of the slow phase exhibited strong dependence on [GdmCl], changing from hundreds of seconds at low denaturant concentration to about 20 ms at 7 M GdmCl. The curvature of this dependence further suggests a complex mechanism of unfolding. Generally, similar kinetics were observed for the trypsinogen.Ca complex. Small differences could be noticed, however, for the fast phase. In agreement, Ca2+ influenced only this stage of the reaction. Analysis of the dependence of the time constant of the fast phase on [CaCl2] indicates that at 4 M GdmCl, trypsinogen.Ca unfolds about sixfold slower than free zymogen, and that native trypsinogen at 4 M GdmCl still exhibits high affinity for Ca2+. Limited data on trypsin unfolding show virtually an identical dependence of the slow phase on [GdmCl]; the fast phase, however was not observed. Moreover, in the 3-4.5 M GdmCl range, a separate phase was detected. It is postulated that this phase is a manifestation of the activation-domain unfolding. The Eyring plots for the fast phase of . trypsinogen and trypsinogen.Ca unfolding are linear, indicating little change in heat capacity for this stage of reaction. The slow step of unfolding, however, shows significant curvature which indicates a substantial increase in heat capacity. Study holds ProTherm entries: 9882 Extra Details: trypsinogen; unfolding kinetics; transition state;,denaturation; guanidinium chloride

Submission Details

ID: sBnWrZ8r

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:39 p.m.

Version: 1

Publication Details
Otlewski J;Sywula A;Kolasinski M;Krowarsch D,Eur. J. Biochem. (1996) Unfolding kinetics of bovine trypsinogen. PMID:9022687
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1AQ7 1997-08-07T00:00:00+0000 2.2 TRYPSIN WITH INHIBITOR AERUGINOSIN 98-B
1AUJ 1997-08-28T00:00:00+0000 2.1 BOVINE TRYPSIN COMPLEXED TO META-CYANO-BENZYLIC INHIBITOR
1AZ8 1997-11-26T00:00:00+0000 1.8 BOVINE TRYPSIN COMPLEXED TO BIS-PHENYLAMIDINE INHIBITOR
1BJU 1998-06-29T00:00:00+0000 1.8 BETA-TRYPSIN COMPLEXED WITH ACPU
1BJV 1998-06-29T00:00:00+0000 1.8 BETA-TRYPSIN COMPLEXED WITH APPU
1BTP 1995-08-11T00:00:00+0000 2.2 UNIQUE BINDING OF A NOVEL SYNTHETIC INHIBITOR, N-[3-[4-[4-(AMIDINOPHENOXY)-CARBONYL]PHENYL]-2-METHYL-2-PROPENOYL]-N-ALLYLGLYCINE METHANESULFONATE TO BOVINE TRYPSIN, REVEALED BY THE CRYSTAL STRUCTURE OF THE COMPLEX
1BTW 1995-05-17T00:00:00+0000 1.7 Episelection: novel KI ~nanomolar inhibitors of serine proteases selected by binding or chemistry on an enzyme surface
1BTX 1995-05-17T00:00:00+0000 1.7 Episelection: Novel Ki ~Nanomolar Inhibitors of Serine Proteases Selected by Binding or Chemistry on an Enzyme Surface
1BTY 1995-05-17T00:00:00+0000 1.5 Crystal structure of beta-trypsin in complex with benzamidine
1BTZ 1995-05-17T00:00:00+0000 2.0 Episelection: novel KI ~nanomolar inhibitors of serine proteases selected by binding or chemistry on an enzyme surface

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Cationic trypsin P00760 TRY1_BOVIN