Year-end Sale % OFF 
Abstract
We studied the temperature‒ and denaturant‒induced denaturation of yeast enolase by means of Fourier transform infrared spectroscopy. The temperature‒induced denaturation/aggregation of the enzyme in the absence of denaturant was highly cooperative and occurred between 55 and 65°C with a midpoint of ~58°C. Above 55°C, the intensity at 1656 cm−1(predominantly α‒helix) decreases as a function of temperature, accompanied by the appearance of two new bands at 1622 and 1696 cm−1, indicating the formation of intermolecular β‒sheet aggregates. Five clearly defined isosbestic points were observed, indicating a two‒state conformational transition. Addition of a non‒denaturing concentration of gdnHCl (0.4 M) caused the thermal denaturation/aggregation of the enzyme to proceed faster, but this revealed no unfolding intermediate. The gdnHCl‒induced unfolding was first detected at a gdnHCl concentration of above 0.4 M, evidenced by loss of α‒helix and β‒sheet structures as functions of denaturant concentration. The fully unfolded state was reached at a gdnHCl concentration of 1.6 M. A significant amount of intermolecular β‒sheet aggregate was detected at gdnHCl concentrations between 0.6 and 1.0 M, which disappeared as the denaturant concentration increased further. The gdnHCl‒unfolded state is a heterogeneous ensemble of turns, helix/loops, and random structures, which continues to change at higher concentrations of denaturant.
Highlights
Enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11) is a glycolic protein that catalyzes the dehydration of 2-phospho-D-glycerate to form phosphoenolpyruvate
At lower temperatures (25–55◦C), yeast enolase exhibits its amide I band maximum at 1655 cm−1, indicating the presence of predominant α-helix structure [23,30]
The amide II band maximum of yeast enolase was observed at 1549 cm−1 at lower temperatures and shifted gradually to 1538 cm−1 during the course of thermal denaturation
Summary
Enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11) is a glycolic protein that catalyzes the dehydration of 2-phospho-D-glycerate to form phosphoenolpyruvate. Binding of metal ions at site I induces conformational changes at the enzyme active site, which enables the binding of substrate or substrate analogue [8]. X-ray crystal structures of yeast enolase in both the apo and holo forms are currently available [10,11]. High-resolution crystal structure revealed that yeast enolase is a modified (βα) barrel protein with a core structure arrangement of ββαα(βα) topology [10,11]. Each monomer of the dimeric enzyme consists of two domains: a smaller N-terminal domain with a three-stranded antiparallel β-meander, followed by a long α-helix and three shorter helices, and a larger C-terminal domain with an 8-fold βα-barrel
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
