Observation of a hybrid random ping-pong mechanism of catalysis for NodST

A mass spectrometry approach

Na Pi, Yonghao Yu, Joseph D. Mougous, Julie A. Leary

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

An efficient enzyme kinetics assay using electrospray ionization mass spectrometry (ESI-MS) was initially applied to the catalytic mechanism investigation of a carbohydrate sulfotransferase, NodST. Herein, the recombinant NodST was overexpressed with a His6-tag and purified via Ni-NTA metal-affinity chromatography. In this bisubstrate enzymatic system, an internal standard similar in structure and ionization efficiency to the product was chosen in the ESI-MS assay, and a single point normalization factor was determined and used to quantify the product concentration. The catalytic mechanism of NodST was rapidly determined by fitting the MS kinetic data into a nonlinear regression analysis program. The initial rate kinetics analysis and product inhibition study described support a hybrid double-displacement, two-site ping-pong mechanism of NodST with formation of a sulfated NodST intermediate. This covalent intermediate was further isolated and detected via trypsin digestion and Fourier transform ion cyclotron resonance mass spectrometry. To our knowledge, these are the first mechanistic data reported for the bacterial sulfotransferase, NodST, which demonstrated the power of mass spectrometry in elucidating the reaction pathway and catalytic mechanism of promising enzymatic systems.

Original languageEnglish (US)
Pages (from-to)903-912
Number of pages10
JournalProtein Science
Volume13
Issue number4
DOIs
StatePublished - Apr 2004

Fingerprint

Catalysis
Mass spectrometry
Mass Spectrometry
His-His-His-His-His-His
Electrospray Ionization Mass Spectrometry
Observation
Electrospray ionization
Cyclotrons
Sulfotransferases
Assays
Enzyme Assays
Fourier Analysis
Affinity Chromatography
Affinity chromatography
Cyclotron resonance
Enzyme kinetics
Trypsin
Digestion
Metals
Regression Analysis

Keywords

  • ESI-MS assay
  • Hybrid random ping-pong mechanism
  • Mass spectrometry
  • NodST

ASJC Scopus subject areas

  • Biochemistry

Cite this

Observation of a hybrid random ping-pong mechanism of catalysis for NodST : A mass spectrometry approach. / Pi, Na; Yu, Yonghao; Mougous, Joseph D.; Leary, Julie A.

In: Protein Science, Vol. 13, No. 4, 04.2004, p. 903-912.

Research output: Contribution to journalArticle

Pi, Na ; Yu, Yonghao ; Mougous, Joseph D. ; Leary, Julie A. / Observation of a hybrid random ping-pong mechanism of catalysis for NodST : A mass spectrometry approach. In: Protein Science. 2004 ; Vol. 13, No. 4. pp. 903-912.
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