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.

scholarly journals Activation of dihydrofolate reductase following thiol modification involves a conformational change at the active site

1998 ◽  
Vol 335 (3) ◽  
pp. 643-646 ◽  
Author(s):  
Ying-Xin FAN ◽  
Zhen-Yu LI ◽  
Li ZHU ◽  
Jun-Mei ZHOU
Keyword(s):  
Kinetic Study ◽  
Conformational Change ◽  
Dihydrofolate Reductase ◽  
Conformational Changes ◽  
Active Site ◽  
Chinese Hamster ◽  
Intrinsic Fluorescence ◽  
Activation Mechanism ◽  
Slow Phase ◽  
Ideal System

Compared with the activation of dihydrofolate reductase (DHFR) by protein denaturants and inorganic salts, activation of the enzyme by thiol modification is relatively slow. Thus it is an ideal system for kinetic study of the activation mechanism. We describe here a kinetic study of the activation of DHFRs from chicken liver and Chinese hamster ovary by p-hydroxymercuribenzoate (p-HMB). The conformational changes in the enzyme molecule that result from the modification were monitored by measuring fluorescence enhancement due to the binding of 2-p-toluidinylnaphthalene-6-sulphonate (TNS), and by monitoring changes in the intrinsic fluorescence of the enzyme. Both activation and the conformational change probed by TNS followed pseudo-first-order kinetics, and the rate constants obtained are in good agreement with each other. The change in intrinsic fluorescence is a biphasic process. The rate of the fast phase, which may reflect a change in the microenvironment of Trp-24 at the active site, coincides with the rate of activation and the conformational change probed by TNS. The rate of the slow phase, which reflects a global conformational change, is about one order of magnitude lower than that of activation. The results indicate that the activation of DHFR by p-HMB is due to modification-induced conformational changes at its active site, rather than the modification of the thiol group itself, which is almost complete within the dead-time of the experiment. This study provides kinetic evidence for the proposal that flexibility at the active site is essential for full expression of catalytic activity.

scholarly journals Inactivation during denaturation of ribonuclease A by guanidinium chloride is accompanied by unfolding at the active site

1995 ◽  
Vol 305 (2) ◽  
pp. 379-384 ◽  
Author(s):  
H J Yang ◽  
C L Tsou
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Ribonuclease A ◽  
Proteinase K ◽  
Peptide Fragments ◽  
Terminal Sequence ◽  
Guanidinium Chloride ◽  
Low Concentrations ◽  
Partial Unfolding ◽  
Absorption Measurements

Inactivation of pancreatic RNAase A occurs in guanidinium chloride (GdmCl) at low concentrations before the unfolding of the molecule as a whole can be detected [Liu and Tsou (1987) Biochim. Biophys. Acta 916, 455-464]. We have now shown that the rate of digestion of the RNAase molecule by either trypsin or proteinase K increases significantly at low concentrations of GdmCl where the enzyme is largely inactivated, but fluorescence and absorption measurements reveal no conformational changes. N-Terminal sequence analysis of the peptide fragments generated shows that proteolysis occurs primarily at or near the active site. The decrease in activity of RNAase at low concentrations of GdmCl is therefore due to partial unfolding of the molecule, particularly at the active site and not to an inhibition by the denaturant.

Conformational Changes in the Active Site Loops of Dihydrofolate Reductase during the Catalytic Cycle†

Biochemistry ◽  
2004 ◽  
Vol 43 (51) ◽  
pp. 16046-16055 ◽  
Author(s):  
Rani P. Venkitakrishnan ◽  
Eduardo Zaborowski ◽  
Dan McElheny ◽  
Stephen J. Benkovic ◽  
H. Jane Dyson ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Conformational Changes ◽  
Active Site ◽  
Catalytic Cycle

scholarly journals Synergistic effects of functionally distinct substitutions in β-lactamase variants shed light on the evolution of bacterial drug resistance

2018 ◽  
Vol 293 (46) ◽  
pp. 17971-17984 ◽  
Author(s):  
Meha P. Patel ◽  
Liya Hu ◽  
Cameron A. Brown ◽  
Zhizeng Sun ◽  
Carolyn J. Adamski ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Conformational Changes ◽  
Active Site ◽  
Enzyme Stability ◽  
Synergistic Effects ◽  
Catalytic Efficiency ◽  
Bacterial Drug Resistance ◽  
Natural Variant ◽  
Loop Conformation ◽  
Shed Light

The CTX-M β-lactamases have emerged as the most widespread extended-spectrum β-lactamases (ESBLs) in Gram-negative bacteria. These enzymes rapidly hydrolyze cefotaxime, but not the related cephalosporin, ceftazidime. ESBL variants have evolved, however, that provide enhanced ceftazidime resistance. We show here that a natural variant at a nonactive site, i.e. second-shell residue N106S, enhances enzyme stability but reduces catalytic efficiency for cefotaxime and ceftazidime and decreases resistance levels. However, when the N106S variant was combined with an active-site variant, D240G, that enhances enzyme catalytic efficiency, but decreases stability, the resultant double mutant exhibited higher resistance levels than predicted on the basis of the phenotypes of each variant. We found that this epistasis is due to compensatory effects, whereby increased stability provided by N106S overrides its cost of decreased catalytic activity. X-ray structures of the variant enzymes in complex with cefotaxime revealed conformational changes in the active-site loop spanning residues 103–106 that were caused by the N106S substitution and relieve steric strain to stabilize the enzyme, but also alter contacts with cefotaxime and thereby reduce catalytic activity. We noted that the 103–106 loop conformation in the N106S-containing variants is different from that of WT CTX-M but nearly identical to that of the non-ESBL, TEM-1 β-lactamase, having a serine at the 106 position. Therefore, residue 106 may serve as a “switch” that toggles the conformations of the 103–106 loop. When it is serine, the loop is in the non-ESBL, TEM-like conformation, and when it is asparagine, the loop is in a CTX-M–like, cefotaximase-favorable conformation.

scholarly journals Conformational Changes in the Active Site Loops of Dihydrofolate Reductase during the Catalytic Cycle

Biochemistry ◽  
2005 ◽  
Vol 44 (15) ◽  
pp. 5948-5948 ◽  
Author(s):  
Rani P. Venkitakrishnan ◽  
Eduardo Zaborowski ◽  
Dan McElheny ◽  
Stephen J. Benkovic ◽  
H. Jane Dyson ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Conformational Changes ◽  
Active Site ◽  
Catalytic Cycle

Protein-induced conformational changes in 16S rRNA during assembly of the Escherichia Coli 30S ribosomal subunit: A STEM study

1987 ◽  
Vol 45 ◽  
pp. 910-911
Author(s):  
M. Boublik ◽  
V. Mandiyan ◽  
J.F. Hainfeld ◽  
J.S. Wall
Keyword(s):  
16S Rrna ◽  
Conformational Changes ◽  
Ribosomal Proteins ◽  
Structural Changes ◽  
Ribosomal Subunit ◽  
Compact Structure ◽  
E Coli ◽  
Freeze Dried ◽  
16S Rna ◽  
30S Subunit

The aim of this study is to understand the mechanism of 16S rRNA folding into the compact structure of the small 30S subunit of E. coli ribosome. The assembly of the 30S E. coli ribosomal subunit is a sequence of specific interactions of 16S rRNA with 21 ribosomal proteins (S1-S21). Using dedicated high resolution STEM we have monitored structural changes induced in 16S rRNA by the proteins S4, S8, S15 and S20 which are involved in the initial steps of 30S subunit assembly. S4 is the first protein to bind directly and stoichiometrically to 16S rRNA. Direct binding also occurs individually between 16S RNA and S8 and S15. However, binding of S20 requires the presence of S4 and S8. The RNA-protein complexes are prepared by the standard reconstitution procedure, dialyzed against 60 mM KCl, 2 mM Mg(OAc)2, 10 mM-Hepes-KOH pH 7.5 (Buffer A), freeze-dried and observed unstained in dark field at -160°.

scholarly journals The Effect of Extraction Conditions on the Barrier and Mechanical Properties of Kefiran Films

Coatings ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 602
Author(s):  
Carmen Rodica Pop ◽  
Teodora Emilia Coldea ◽  
Liana Claudia Salanţă ◽  
Alina Lăcrămioara Nistor ◽  
Andrei Borşa ◽  
...  
Keyword(s):  
Water Vapor ◽  
Conformational Changes ◽  
Water Vapor Permeability ◽  
Edible Films ◽  
Film Structure ◽  
Water Soluble ◽  
Vapor Permeability ◽  
Water Molecules ◽  
Compact Structure ◽  
Technology Applications

Kefiran is an exopolysaccharide classified as a heteropolysaccharide comprising glucose and galactose in equimolar quantities, and it is classified as a water-soluble glucogalactan. This work aimed to investigate the effect of different extraction conditions of kefiran on the structural and physical properties of the edible films obtained. Fourier-transform infrared spectroscopy and scanning electron microscopy were performed, together with a determinations of moisture content, solubility, water vapor permeability and degree of swelling. The kefiran films presented values of the water vapor permeability between 0.93 and 4.38 × 10−11 g/m.s.Pa. These results can be attributed to the development of a more compact structure, where glycerol had no power to increase the free volume and the water vapor diffusion through their structure. The possible conformational changes in the kefiran film structure, due to the interspersing of the plasticizers and water molecules that they absorb, could be the reason for producing flexible kefiran films in the case of using glycerol as a plasticizer at 7.5% w/w. Moreover, it was observed that the extraction conditions are a significant factor in the properties of these films and their food technology applications.

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