Finding the Entrance to an Enzyme Active Site: Lipoxygenase

• Other Authors: Bradshaw, Miles D. (Miles David) (authoraut)
• Format: Others
• Language: English; English
• Published: Florida State University
• Subjects: Biochemistry
• Online Access: http://purl.flvc.org/fsu/fd/FSU_migr_etd-9295

The relationship between protein structure and the specificity of substrate oxidation remains a key problem in the study of the lipoxygenase enzyme family. Polyunsaturated lipid substrates reach the non-heme iron contained within the active site through a bent channel, and the entrance to this channel was defined by the application of spin-labeling. Spin-labels were engineered at five positions surrounding the active site, and the entrance to the substrate channel was defined by pulsed-EPR distance measurements between these sites and a bound choline spin-labeled lysolecithin substrate analog. The polar end of this lipid was located at the surface of the enzyme, at the interface of helices-2 and -11. Differences in the structure of helix-2 have been discussed in the literature as possible sources of biochemical variability among lipoxygenases including control of the position of substrate oxidation and product stereochemistry, but the role this solvent exposed helix plays in enzymatic function is poorly understood. Helix-2 in soybeans contains a single π-helical turn in the center. A spin-label scan of 18 / 21 residues of helix-2 shows that the π-helical region, in solution, has unusual dynamics under conditions favorable for catalysis of lipid substrates (pH 9 or added lipid). The backbone of this segment undergoes fluctuation on the nanosecond timescale, while the regions before and after appear to undergo smaller changes in motion under these conditions. EPR spectra of frozen samples revealed a decrease in polarity / proticity in response to an increase in pH from 7 to 9, especially within the π-helical segment of helix-2. Power saturation measurements demonstrate that this decrease in polarity caused an enhancement in the intrinsic spin-relaxation rates. Distance dependent interactions between spin-label sites on helix-2 and the high spin (S = 4/2) Fe2+ contained within resting enzyme were also observed at pH 7. Upon lipid binding, power saturation experiments suggest that the coordination state of iron alters the rate of magnetic interaction with the spin-labels on helix-2. A salt-bridge interaction between H248 and E256 on helix-2 was identified as limiting substrate access at pH 7, and affecting the dynamics of helix-2. Overall, the results show that flexibility of the middle region of helix-2 (residues 261-267) is necessary for substrate to enter substrate channel from a surface entrance located near helices-2 and -11. === A Dissertation submitted to the Department of Biological Sciencein partial fulfillment of therequirements for the degree ofDoctor of Philosophy. === Spring Semester, 2015. === March 23, 2015. === Enzymes, EPR, Lipids, Lipoxygenase, Magnetic Resonance, Spin-labeling === Includes bibliographical references. === Betty J. Gaffney, Professor Directing Dissertation; Timothy A. Cross, University Representative; Michael Blaber, Committee Member; Darin R. Rokyta, Committee Member; Hengli Tang, Committee Member.