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 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

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.

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.

Conformational changes in the catalytic cycle of protochlorophyllide oxidoreductase: what lessons can be learnt from dihydrofolate reductase?

2009 ◽  
Vol 37 (2) ◽  
pp. 354-357 ◽  
Author(s):  
Derren J. Heyes ◽  
Nigel S. Scrutton
Keyword(s):  
Dihydrofolate Reductase ◽  
Conformational Changes ◽  
Catalytic Mechanism ◽  
Structural Information ◽  
Chlorophyll Biosynthesis ◽  
Catalytic Cycle ◽  
Protochlorophyllide Oxidoreductase ◽  
Protein Motions ◽  
Kinetic Information ◽  
Related Enzyme

In chlorophyll biosynthesis, the light-activated enzyme, POR (protochlorophyllide oxidoreductase), has been shown to be an excellent model system for studying the role of protein motions during catalysis. The catalytic cycle of POR is understood in detail and comprises an initial photochemical reaction, which is followed by a number of ‘dark’ steps. The latter steps in the reaction cycle have been shown to involve a series of ordered product release and substrate rebinding events and are known to require conformational changes in the protein in order to proceed. However, owing to the current lack of any structural information on the enzyme, the nature of these conformational rearrangements remains poorly understood. By contrast, there is a wealth of structural and kinetic information available on the closely related enzyme dihydrofolate reductase, which is known to have a similar catalytic mechanism to POR. Dihydrofolate reductase is able to adopt an ‘occluded’ and a ‘closed’ structure, depending on which ligand is bound in the active site, and as a result, the catalytic cycle is controlled by a ‘switching’ between these two conformations. By analogy, we suggest that a similar cycling between different conformations may be operating in POR.

scholarly journals Active site lysine backbone undergoes conformational changes in the bacteriorhodopsin photocycle.

1994 ◽  
Vol 269 (10) ◽  
pp. 7387-7389
Author(s):  
H. Takei ◽  
Y. Gat ◽  
Z. Rothman ◽  
A. Lewis ◽  
M. Sheves
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Bacteriorhodopsin Photocycle
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