Reconstitution of the Holoenzyme Form of Escherichia coli Porphobilinogen Deaminase from Apoenzyme with Porphobilinogen and Preuroporphyrinogen: A Study Using Circular Dichroism Spectroscopy

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• 作者：Sarah J. Awan ; Giuliano Siligardi ; Peter M. Shoolingin-Jordan ; and Martin J. Warren
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：July 29, 1997
• 年：1997
• 卷：36
• 期：30
• 页码：9273 - 9282
• 全文大小：490K
• 年卷期：v.36,no.30(July 29, 1997)
• ISSN：1520-4995

文摘

Porphobilinogen deaminase (PBG-D), an early enzyme ofthe tetrapyrrole biosynthetic pathway,catalyzes the formation of a tetrapyrrole chain, preuroporphyrinogen,from four molecules of porphobilinogen (PBG). The PBG-D apoenzyme is responsible for theautocatalytic synthesis and covalent attachmentof a dipyrromethane cofactor at its active site. In this paper anefficient method for the purification ofEscherichia coli PBG-D apoenzyme using an affinitychromatography resin is reported. Circular dichroism(CD) spectra of apoenzyme and holoenzyme were recorded and significantdifferences in both the backboneand aromatic region of the spectra were observed. The differencesin the spectra allowed the reconstitutionof holoenzyme from purified apoenzyme with PBG and preuroporphyrinogenin solution to be monitoredseparately by CD. Apoenzyme incubated with preuroporhyrinogen gavea CD spectrum that was muchmore like the CD spectrum of holoenzyme than apoenzyme incubated withPBG. The results showedclearly that the cofactor was generated much more rapidly frompreuroporphyrinogen than from PBG.Changes in the CD spectrum associated with the aromatic side-chainregion, in particular the contributionassigned to phenylalanine-62, were found to correlate well with theactivity of the reconstituted enzyme.Phenylalanine-62 is located in close proximity to the cofactor andacts as a sensitive probe to active-sitechanges. The stability of the holoenzyme and apoenzyme werecompared with respect to both heat andsusceptibility to proteolysis. The results were consistent with amodel for the apoenzyme in which, inthe absence of the cofactor, the three domains of the protein are heldless rigidly together, thereby makingthe protein more susceptible to heat denaturation and proteolysis.The CD spectrum of the holoenzymewas found to be similar at both pH 5.1 and 7.4, suggesting that thecrystal structure, determined at pH5.1, is likely to be similar at physiological pH values.