January 2012

Combining Tools to Understand Enzyme Function

New insights into metal ions at an enzyme’s active site.

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Image © 2012 American Chemical Society.

Reprinted with permission from Daughtry, K.D., et al., “Quaternary ammonium oxidative demethylation: X-ray crystallographic, resonance Raman, and UV-Visible spectroscopic analysis of a Rieske-type demethylase” (2012) Journal of the American Chemical Society, 134 (5), pp 2823–2834. [DOI: 10.1021/ja2111898]

The Science

A new technology has been developed that simultaneously carries out crystallography and UV-visible and Raman spectroscopy to determine the atomic structure of the entire protein, and electronic and vibrational structures of the metal ions or cofactors within.

The Impact

This new combined technology will provide information about the oxidation state of metal ions at an enzyme’s active site which is important for understanding enzyme behavior.

Summary

Structure and function are intimately linked but do not necessarily predict the other. For example, X-ray crystallography provides 3-D atomic structural information about biological macromolecules but does not define important details about metal ions. However, the oxidation state of metal ions at an enzyme’s active site has a critical effect on enzyme behavior. Thus, an enzyme’s catalytic function derives from the electronic structure of those atoms influencing or directly participating in the reaction, information not revealed by the scattering methods used in X-ray crystallography. The combined instrumentation has been used to study the process of demethylation of an organic substrate molecule by an enzyme whose active site includes an iron-sulfur cluster. The authors used spectroscopy to follow the change in the oxidation state of the cluster during the crystallography data collection, and to formulate a mechanism for the process. The results provide insight into an important class of phenomena that control cellular behavior. The technology was developed by scientists at the Protein Crystallography Research Resource at the National Synchrotron Light Source at Brookhaven National Laboratory. The new study was led by Allen M. Orville of Brookhaven and Pinghua Liu and Karen N. Allen of Boston University.

Contact

Dr. Allen Orville
Brookhaven National Laboratory
amorv@bnl.gov

Funding

Basic Research: DOE Office of Science, Biological and Environmental Research program; National Synchrotron Light Source: DOE Office of Science, Basic Energy Sciences program

Publications

Daughtry, K.D., et al., “Quaternary ammonium oxidative demethylation: X-ray crystallographic, resonance Raman, and UV-Visible spectroscopic analysis of a Rieske-type demethylase” (2012) Journal of the American Chemical Society, 134 (5), pp 2823–2834. [DOI: 10.1021/ja2111898]

Highlight Categories

Program: BES, SUF, BER, BSSD

Performer/Facility: University, DOE Laboratory, SC User Facilities, BES User Facilities, NSLS

Last modified: 9/3/2013 12:11:23 PM