Sequential conformation change and activation of chicken liver dihydrofolate reductase in low concentration of guanidine hydrochloride

1997 ◽  
Vol 40 (2) ◽  
pp. 202-209
Author(s):  
Yingxin Fan ◽  
Li Zhu ◽  
Junmei Zhou ◽  
Chenglu Zou
Keyword(s):  
Dihydrofolate Reductase ◽  
Guanidine Hydrochloride ◽  
Chicken Liver ◽  
Conformation Change ◽  
Low Concentration

scholarly journals Activation of chicken liver dihydrofolate reductase by urea and guanidine hydrochloride is accompanied by conformational change at the active site

1996 ◽  
Vol 315 (1) ◽  
pp. 97-102 ◽  
Author(s):  
Ying-xin FAN ◽  
Ming JU ◽  
Jun-mei ZHOU ◽  
Chen-lu TSOU
Keyword(s):  
Catalytic Activity ◽  
Dihydrofolate Reductase ◽  
Conformational Changes ◽  
Active Site ◽  
Guanidine Hydrochloride ◽  
Chicken Liver ◽  
Peptide Fragments ◽  
Enhanced Fluorescence ◽  
Compact Structure ◽  
Mouse Leukaemia

It has been reported that the activation of dihydrofolate reductase (DHFR) from L1210 mouse leukaemia cells by KCl or thiol modifiers is accompanied by increased digestibility by proteinases [Duffy, Beckman, Peterson, Vitols and Huennekens (1987) J. Biol. Chem. 262, 7028–7033], suggesting a loosening up of the general compact structure of the enzyme. In the present study, the peptide fragments liberated from the chicken liver enzyme by digestion with trypsin in dilute solutions of urea or guanidine hydrochloride (GuHCl) have been separated by FPLC and sequenced. The sequences obtained are unique when compared with the known sequence of DHFR and thus allow the points of proteolytic cleavage identified for the urea- and GuHCl-activated enzyme to be at or near the active site. It was also indicated by the enhanced fluorescence of 2-p-toluidinylnaphthalene 6-sulphonate that conformational changes at the active site in dilute GuHCl parallel GuHCl activation. The above results indicate that the activation of DHFR in dilute denaturants is accompanied by a loosening up of its compact structure especially at or near the active site, suggesting that the flexibility at its active site is essential for the full expression of its catalytic activity.

A structure—function study of dihydrofolate reductase by protein engineering

1986 ◽  
Vol 317 (1540) ◽  
pp. 405-413 ◽  
Keyword(s):  
Structure Function ◽  
Dihydrofolate Reductase ◽  
Conformational Changes ◽  
Guanidine Hydrochloride ◽  
Disulphide Bond ◽  
E Coli ◽  
Transition State Stabilization ◽  
Disulphide Bond Formation ◽  
High Resolution Structure ◽  
Charge Interaction

Several mutants of the enzyme dihydrofolate reductase (DHFR) have been engineered by oligonucleotide-directed mutagenesis of the cloned E. coli gene. The mutations were designed to address specific questions about DHFR structure-function relations that arose from the analysis of the high-resolution structure. Mutations at the active site have revealed that the invariant residue aspartate-27 is involved in substrate protonation, and not in transition-state stabilization as previously thought. The 2.0 Å (1 Å = 10 -1 nm = 10 -10 m) refined structures of the Asn-27 and Ser-27 mutant enzymes reveal that the enhanced binding observed for the 2,4-diamino pteridine and pyrimidine inhibitors is probably not attributable to the charge interaction between Asp-27 and a protonated N-1 of the inhibitor. Substitution of a cysteine for a proline at position 39 places two sulphydryls within bonding distance, and under certain oxidation conditions they will quantitatively form a disulphide bond. The refined 2.0 Å structures of both reduced and oxidized forms of this mutant show that only minor conformational changes occur for disulphide bond formation. The crosslinked enzyme is significantly more conformationally stable to denaturants such as guanidine hydrochloride and urea.

Primary structure of chicken liver dihydrofolate reductase

Biochemistry ◽  
1980 ◽  
Vol 19 (4) ◽  
pp. 667-678 ◽  
Author(s):  
A. Ashok Kumar ◽  
Dale T. Blankenship ◽  
Bernard T. Kaufman ◽  
James H. Freisheim
Keyword(s):  
Dihydrofolate Reductase ◽  
Primary Structure ◽  
Chicken Liver
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