Motional dynamics of the catalytic loop in OMP synthase

Gary P. Wang, Sean M. Cahill, Xiaohong Liu, Mark E. Girvin, Charles Grubmeyer

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

In de novo pyrimidine biosynthesis, orotate phosphoribosyltransferase catalyzes the formation of orotidine 5'-monophosphate (OMP) from orotic acid and α-D-5-phosphoribosyl-1-pyrophosphate (PRPP). The known three- dimensional structure of the dimeric enzyme from Salmonella typhimurium is similar to that of other Type I phosphoribosyltransferases (nucleotide synthases) with a solvent-exposed active site atop a Rossman-type nucleotide binding fold. The three-dimensional structure of an enzyme - inhibitor complex [Henriksen et al. (1996) Biochemistry 35, 3803-3809] indicates that of the two identical solvent-exposed loops can descend to cover the active site of the adjacent subunit of the dimeric enzyme. Catalytically essential residues are known to reside on this loop. In the present work, sensitivity toward limited proteolysis by trypsin confirms that the loop is solvent- exposed. Protection by PRPP and, to a lesser extent, by OMP demonstrates the existence of a second, trypsin-inaccessible, loop position. Two-dimensional 1H-15N NMR relaxation experiments on [α-15N]histidine-labeled WT OPRTase yielded backbone 15N T1 and T2 relaxation times and 15{1H} NOE for His-105 (a loop residue) that are characteristic of small peptides. These results document that the surface loop is highly flexible in the unliganded enzyme. Addition of a hydrolytically stable PRPP analogue to the enzyme resulted in a significant reduction of His-105 peak intensity, indicating a dramatic change in the dynamic properties of the loop backbone in the analogue-ligated enzyme. 1H NMR titrations on histidine C2 protons, coupled with 1H and 31P titrations monitoring the C1H and 5-phosphate PRPP resonances, allowed the quantitation of the rates of loop movement during product release, and relate protein motion to enzymatic catalysis. These results suggest that loop opening and PRPP release is a two-step process, whose overall rate is partially rate-limiting in the reverse pyrophosphorolysis reaction.

Original languageEnglish (US)
Pages (from-to)284-295
Number of pages12
JournalBiochemistry
Volume38
Issue number1
DOIs
StatePublished - Jan 5 1999

Fingerprint

Phosphoribosyl Pyrophosphate
Enzymes
Titration
Histidine
Trypsin
Catalytic Domain
Orotate Phosphoribosyltransferase
Nucleotides
Nuclear magnetic resonance
Orotic Acid
Proteolysis
Biochemistry
Salmonella
Biosynthesis
Enzyme Inhibitors
Salmonella typhimurium
Catalysis
Relaxation time
Protons
Phosphates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Wang, G. P., Cahill, S. M., Liu, X., Girvin, M. E., & Grubmeyer, C. (1999). Motional dynamics of the catalytic loop in OMP synthase. Biochemistry, 38(1), 284-295. https://doi.org/10.1021/bi982057s

Motional dynamics of the catalytic loop in OMP synthase. / Wang, Gary P.; Cahill, Sean M.; Liu, Xiaohong; Girvin, Mark E.; Grubmeyer, Charles.

In: Biochemistry, Vol. 38, No. 1, 05.01.1999, p. 284-295.

Research output: Contribution to journalArticle

Wang, GP, Cahill, SM, Liu, X, Girvin, ME & Grubmeyer, C 1999, 'Motional dynamics of the catalytic loop in OMP synthase', Biochemistry, vol. 38, no. 1, pp. 284-295. https://doi.org/10.1021/bi982057s
Wang, Gary P. ; Cahill, Sean M. ; Liu, Xiaohong ; Girvin, Mark E. ; Grubmeyer, Charles. / Motional dynamics of the catalytic loop in OMP synthase. In: Biochemistry. 1999 ; Vol. 38, No. 1. pp. 284-295.
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