HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 278, Issue 9, 6921-6927, February 28, 2003

This Article Full Text Full Text (PDF) All Versions of this Article: 278/9/6921    most recent M211111200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Grabner, G. K. Articles by Switzer, R. L. Search for Related Content PubMed PubMed Citation Articles by Grabner, G. K. Articles by Switzer, R. L. Social Bookmarking                      What's this?

Kinetic Studies of the Uracil Phosphoribosyltransferase Reaction Catalyzed by the Bacillus subtilis Pyrimidine Attenuation Regulatory Protein PyrR^*

From the Department of Biochemistry, University of Illinois, Urbana, Illinois 61801

The PyrR protein from Bacillus subtilis and many other bacteria is a bifunctional protein. Its primary function is the regulation of expression of pyrimidine biosynthetic (pyr) genes by binding to specific sites on pyr mRNA in a uridine nucleotide-dependent manner and altering the folding of downstream RNA to promote termination of transcription. PyrR also catalyzes the uracil phosphoribosyltransferase (UPRTase) reaction even though it bears little amino acid sequence similarity to other bacterial UPRTases. The PyrR-catalyzed UPRTase reaction obeyed a Ping Pong steady state kinetic pattern under all conditions examined, but no catalysis of [¹⁴C]uracil-UMP and [³²P]PP_i-phosphoribosylpyrophosphate exchange reactions could be detected. Steady state equations for Ordered Bi Bi mechanisms for PyrR that include a kinetically irreversible conformational change after binding of PRPP but before uracil binding were shown to account for the Ping Pong pattern of the enzyme. This mechanism was supported by the following experimental observations. The reverse reaction was extremely slow with a catalytic rate constant 3300 times smaller than for the forward reaction. Patterns of product inhibition of the forward reaction were consistent with a version of the irreversible conformational change model in which PyrR returns to the unliganded conformation before dissociation of UMP and were inconsistent with several other kinetic mechanisms. UMP and phosphoribosylpyrophosphate were shown by equilibrium dialysis to bind to free PyrR (dissociation constants of 27 ± 3 and 18 ± 2 µM, respectively), but uracil and PP_i did not bind at equilibrium concentrations up to 750 µM. We propose that the conformational change kinetic model developed for PyrR can also account for numerous other reports of Ping Pong kinetics for various phosphoribosyltransferases that do not form the phosphoribosyl-enzyme intermediate predicted by classic Ping Pong kinetics.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				C. L. Turnbough Jr. and R. L. Switzer Regulation of Pyrimidine Biosynthetic Gene Expression in Bacteria: Repression without Repressors Microbiol. Mol. Biol. Rev., June 1, 2008; 72(2): 266 - 300. [Abstract] [Full Text] [PDF]

				P. Chander, K. M. Halbig, J. K. Miller, C. J. Fields, H. K. S. Bonner, G. K. Grabner, R. L. Switzer, and J. L. Smith Structure of the Nucleotide Complex of PyrR, the pyr Attenuation Protein from Bacillus caldolyticus, Suggests Dual Regulation by Pyrimidine and Purine Nucleotides J. Bacteriol., March 1, 2005; 187(5): 1773 - 1782. [Abstract] [Full Text] [PDF]