Biochemistry zyxwvu 1986, 25, zyxwvu 905-912 905 Biosynthesis of Porphyrins and Corrins. zyxw 2. Isolation, Purification, and NMR Investigations of the Porphobilinogen-Deaminase Covalent Complexf Jeremy N. S. Evans,t Gerard0 Burton,* Paul E. Fagerness, Neil E. Mackenzie, and A. Ian Scott* Received June 26, 1985 Center for Biological NMR. Department of Chemistry, Texas A&M University, College Station, Texas 77843 ABSTRACT: The procedures for the generation of enzyme-substrate complexes from labeled porphobilinogens ([2,1 1-13C]PBG and [2,6,1 1-3H]PBG) with deaminase and the methods employed for their purification are described. Use of 13C NMR failed to detect the substrate bound to the enzyme, suggesting that the line width must be inordinately large. The complex was found to disproportionate with time when stored at 25 OC. However, enzyme-bound uroporphyrinogen I (uro’gen I) was detected, both in the intact protein and in the oligopeptides from tryptic digestion and peptide mapping. The first detection of an enzyme- substrate complex by 3H N M R is described for [3H]PBG and deaminase. The line widths of the observed resonances were found to be extremely large and dependent upon temperature, giving chemical shifts that suggest the involvement of a sulfhydryl group as the nucleophilic enzyme group that binds the substrate. The catalytic competence of this complex was also demonstrated by displacing bound [3H]PBG with unlabeled PBG. During the resultant formation of [3H]uro’gen I, a transient low-intensity signal was detected that has been tentatively assigned to the highly reactive azafulvene species, proposed in several mechanistic schemes for porphyrin biosynthesis. T e enzyme porphobilinogen (PBG) deaminase (EC 4.3.1.8)* catalyzes the head-to-tail condensation of 4 mol of the monopyrrole porphobilinogen (PBG, 1) to preuro- porphyrinogen (pre-uro’gen), whose release and stabilization as (hydroxymethy1)bilane (HMB, 7) has been the subject of extensive investigation [see preceding paper (Evans et al., 1986)l. HMB may cyclize chemically to uro’gen I or serve as the substrate for uro’gen I11 cosynthase (EC 4.2.1.75) to form uro’gen I11 (Scheme I). The mechanism by which PBG deaminase polymerizes PBG and the structure of the true product of the enzyme have been the subject of much speculation [see preceding paper and reviewed in Buckley (1977) and Akhtar zyxwvutsr & Jordan (1978)l but to date has remained unclear. The order of assembly of the four units was investigated independently by Battersby et al. (1979a,b, 1983a,b), Jordan and Seehra (1979), and Seehra and Jordan (1980). Both groups established that the order of assembly is the expected one: ring A, followed by rings B, C, and D. The discovery by Anderson and Desnick (1980) that human deaminase forms stable covalent complexes bearing up to four condensed PBG units stimulated studies on the enzyme-substrate (ES) complexes from Rhodopseu- domonas spheroides (Jordan & Berry, 1981; Berry & Jordan, 1981; Berry et al., 1981), Euglena gracilis (Williams et al., 1981; Battersby et al., 1983a,b), and rat spleen (Williams, 1984). Studies on the R . spheroides ES complex by Jordan and Berry (1981) suggested that the substrate is covalently attached to the enzyme, and further work by Berry and Jordan (1981) and Berry et al. (1981) identified the existence of four bands by gel electrophoresis. Using [14C]PBG, Berry et al. elegantly showed that the bands corresponded to native enzyme and ES complexes bearing one, two, and three bound PBG Supported by the National Institutes ofHealth (AM32034) and the *Present address: Department of Chemistry, Massachusetts Institute Present address: Departamento de Quimica Organica, Universidad Robert A. Welch Foundation. of Technology, Cambridge, MA 02139. de Buenos Aires, Buenos Aires, Argentina. 0006-2960 zyxwvuts f 86 f 0425-0905$01.50 f zyxwvutsr 0 units. Interestingly, unlike the human enzyme, no complex corresponding to bound tetrapyrrole was detected. The identification and characterization of such ES com- plexes and the discovery of their unusual stability3suggest that it may be possible to probe the structure of the active site and the nature of binding ofithe substrate. A suitable technique for this purpose is NMR spectroscopy, and in particular 13C NMR spectroscopy, the power of which has already been established [reviewed in Mackenzie et al. (1984)l in defining the structure and stereochemistry of enzyme-inhibitor and enzyme-substrate complexes with proteases of modest mo- lecular weight (<30000). In this paper, we describe the use of I3C NMR and tritium (3H) NMR spectroscopy in an at- tempt to elucidate the structure and mechanism of PBG de- aminase. EXPERIMENTAL PROCEDURES Materials. All reagents employed were of the highest grade obtainable. [14C]- and [13C]PBG were prepared by the methods outlined in the preceding paper. [3H]PBG was prepared by similar methods from [ 3H]aminolevulinic acid (ALA) (Evans et al., 1985). Trypsin (TPCK treated), Pronase E, and polyacrylamide gel electrophoresis reagents were ob- tained from Sigma Chemical Co. All enzyme manipulations were carried out at 4 OC except where stated otherwise. Purification zyxwv of PBG Deaminase. Deaminase for enzyme- substrate NMR investigations was prepared from ca. 3 kg wet Abbreviations: PBG, porphobilinogen; NMR, nuclear magnetic resonance; pre-uro’gen, preuroporphyrinogen; HMB, (hydroxymethy1)- bilane; uro’gen I, uroporphyrinogen I; uro’gen 111, uroporphyrinogen 111; ALA, aminolevulinic acid; DEAE, diethylaminoethyl; Tris, tris(hydrox- ymethy1)aminomethane; FID, free induction decay. IUB has recently recommended the name hydroxymethylbilane (HMB) synthase, but as is clear from the work presented here, this name no better represents the enzyme’s function than does the old systematic name uroporphyrinogen I synthase. However, the name porphobilinogen deaminase is noncommittal as to the nature of the product of the enzyme and is therefore employed in this paper. The stability of the ES complexes at high protein concentration is a point that we dispute in this paper. zyxw 0 1986 American Chemical Society