Ligand-induced changes in the conformational stability of bovine trypsinogen and their implications for the protein function.


Abstract

Bovine trypsinogen was used as a model protein for studying changes in the conformational stability induced by pH or binding of the calcium ion. Spectrophotometrically monitored thermal unfolding of trypsinogen and beta-trypsin in the acidic pH range yielded substantial differences in the stability parameters. Compared to beta-trypsin, trypsinogen exhibits lower enthalpy of denaturation delta Hden, higher denaturational heat capacity change delta Cp,den, but very similar temperature of denaturation Tden. pH-dependence of the conformational stability of the ligand-free trypsinogen, measured also by GdnCl-induced unfolding, is bell shaped with the maximum free energy of unfolding delta Gden = 10.9 kcal/mole at pH 5.5 (4.5 pH units below its isoelectric point). At pH 8.3 the conformational stability of the zymogen drops to delta Gden = 3.2 kcal/mole, but increases by delta delta Gden = 6.1 kcal/mole in the presence of Ca2+. This significant stabilization of the zymogen by the calcium ion is also pH-dependent. To assess the effect of Ca2+ on the trypsinogen molecule, the spectrophotometric titrations and NOESY spectra were carried out. Based on the structural analysis, the long range effects between Ca2+-->Ile73-->Trp141 and the interdomain His40-Asp194 ion pair are proposed to be partially responsible for trypsinogen stabilization. Additionally, the steady-state parameters for hydrolysis of the oligopeptide amide substrate catalysed by free trypsinogen, its complexes with Ca2+ and the IleVal dipeptide and by beta-trypsin were measured. It appears that in the pH range 5.5 to 8.3 the stability and the catalytic activity/ligand binding properties are fully separated. Whereas the deprotonation of His57 accounts for the increase of kcat/km parameter, deprotonation of His40 is involved in the huge decrease of the conformational stability. Similarly, a large stabilization by the calcium ion is not accompanied by changes in enzymatic activity. Presented data are encouraging for an enzyme design directed toward improved stability. Study holds ProTherm entries: 7485, 7486, 7487, 7488, 7489 Extra Details: trypsinogen; stability change; calcium; enzyme catalysis;,ligand binding

Submission Details

ID: TxL2hXBj3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:34 p.m.

Version: 1

Publication Details
Bulaj G;Otlewski J,J. Mol. Biol. (1995) Ligand-induced changes in the conformational stability of bovine trypsinogen and their implications for the protein function. PMID:7723025
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
100.0 Anionic trypsin Q29463 TRY2_BOVIN