Kinetics of biotinyl-5'-adenylate synthesis catalyzed by the Escherichia coli repressor of biotin biosynthesis and the stability of the enzyme-product complex.
The Escherichia coli repressor of biotin biosynthesis is both a biotin ligase and the repressor of transcriptional initiation at the biotin biosynthetic operon. The small molecule, biotinyl-5'-adenylate (bio-5'-AMP), is the intermediate in the biotin ligation reaction and the positive allosteric effector for sequence-specific DNA binding by BirA. Synthesis of the adenylate from the substrates biotin and ATP is catalyzed by BirA. Although BirA and other biotin holoenzyme synthetases have been the subject of biochemical studies, no direct measurements of the bio-5'-AMP synthesis reaction have been reported. No information relating to the mechanism and kinetic parameters governing adenylate synthesis is available. In addition to this lack of kinetic information, the thermodynamic stability of the BirA-bio-5'-AMP complex is not known. Since the BirA-adenylate complex plays a pivotal role in the biotin regulatory system, both the kinetic and thermodynamic information are essential to a quantitative understanding of the system. We have developed a method for measuring the time course of bio-5'-AMP synthesis. The results of these measurements indicate that the time course is characterized by an initial burst followed by a slow linear phase. The burst corresponds to the rapid synthesis of 1 mol of product per mole of enzyme, and the rate of the slow linear phase is limited by the release of product from the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Xu
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Department of Chemistry and Biochemistry, University of Maryland, Baltimore 21228.
Beckett
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D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001710:Biotin / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000737:chemistry; D019731:Carbon-Nitrogen Ligases; D002384:Catalysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007700:Kinetics; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics; D014157:Transcription Factors
C082099:5'-AMP-biotin; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D000249:Adenosine Monophosphate; D001710:Biotin; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli