1FUR

FUMARASE MUTANT H188N WITH BOUND SUBSTRATE L-MALATE AT PUTATIVE ACTIVATOR SITE

Summary for 1FUR

• Entry DOI: 10.2210/pdb1fur/pdb
• Descriptor: FUMARASE C, D-MALATE (3 entities in total)
• Functional Keywords: hydrolyase, carbon oxygen lyase, kreb's cycle enzyme, fumarate hydratase
• Biological source: Escherichia coli
• Cellular location: Cytoplasm: P05042
• Total number of polymer chains: 2
• Total formula weight: 101317.63

Authors

Weaver, T.M.,Lees, M.,Banaszak, L.J. (deposition date: 1997-01-09, release date: 1997-07-23, Last modification date: 2024-05-22)

Primary citation

Weaver, T.,Lees, M.,Banaszak, L.
Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site.
Protein Sci., 6:834-842, 1997

PubMed Abstract: Two mutant forms of fumarase C from E. coli have been made using PCR and recombinant DNA. The recombinant form of the protein included a histidine arm on the C-terminal facilitating purification. Based on earlier studies, two different carboxylic acid binding sites, labeled A- and B-, were observed in crystal structures of the wild type and inhibited forms of the enzyme. A histidine at each of the sites was mutated to an asparagine. H188N at the A-site resulted in a large decrease in specific activity, while the H129N mutation at the B-site had essentially no effect. From the results, we conclude that the A-site is indeed the active site, and a dual role for H188 as a potential catalytic base is proposed. Crystal structures of the two mutant proteins produced some unexpected results. Both mutations reduced the affinity for the carboxylic acids at their respective sites. The H129N mutant should be particularly useful in future kinetic studies because it sterically blocks the B-site with the carboxyamide of asparagine assuming the position of the ligand's carboxylate. In the H188N mutation at the active site, the new asparagine side chain still interacts with an active site water that appears to have moved slightly as a result of the mutation.

PubMed: 9098893

Experimental method

X-RAY DIFFRACTION (1.95 Å)