Protein Science
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Protein Science (2004), 13:903-912. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pi, N.
Right arrow Articles by Leary, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pi, N.
Right arrow Articles by Leary, J. A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Observation of a hybrid random ping-pong mechanism of catalysis for NodST: A mass spectrometry approach

Na Pi1, Yonghao Yu1, Joseph D. Mougous2,3 and Julie A. Leary1

1 Department of Chemistry,
2 Department of Molecular Cell Biology and
3 Howard Hughes Medical Institute, University of California (UC), Berkeley, California 94720, USA

(RECEIVED December 17, 2003; FINAL REVISION January 22, 2004; ACCEPTED January 22, 2004)



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.

Keywords: NodST; mass spectrometry; ESI-MS assay; hybrid random ping-pong mechanism

Abbreviations: {Delta}Di-6S, {alpha}-{Delta}UA-[1->3]-GalNAc-6S • 3'PB motif, 3'-phophate binding motif • 5'PSB-loop, 5'-phosphosulfate binding loop • APS, adenosine 5'-phosphosulfate • ASST, arylsulfate sulfotransferase • ATP, adenosine 5'-triphosphate • ER, estrogen receptor • ESI-MS, electrospray ionization mass spectrometry • EST, estrogen sulfotransferase • FST, flavonol sulfotransferase • FT-ICR MS, Fourier transform ion cyclotron resonance mass spectrometry • GalNAc, N-acetylgalactosamine • GlcNAc, N-acetylglucosamine • HNDST, N-deacetylase/N-sulfotransferase • NDP, nucleotide diphosphate • Ni-NTA, nickel-nitrilotriacetic acid • PAP, 3'-phosphoadenosine 5'-phosphate • PAPS, 3'-phosphoadenosine 5'-phosphosulfate • PST, phenol sulfotransferase • SIM, selected ion monitoring

Reprint requests to: Julie A. Leary, Department of Chemistry, University of California, Berkeley, CA 94720, USA; e-mail: leary{at}socrates.berkeley.edu; fax: (510) 642-9295.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03581904.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
N. Pi, C. L. F. Meyers, M. Pacholec, C. T. Walsh, and J. A. Leary
Mass spectrometric characterization of a three-enzyme tandem reaction for assembly and modification of the novobiocin skeleton
PNAS, July 6, 2004; 101(27): 10036 - 10041.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Copyright © 2004 by The Protein Society.