The primary structure of the alpha subunit of protocatechuate 3,4-dioxygenase. I. Isolation and sequence of the tryptic peptides.

The carboxymethylated alpha subunit of protocatechuate 3,4-dioxygenase was digested with trypsin. The 14 tryptic peptides were isolated by ion exchange chromatography on DEAE-Sephadex and by gel filtration chromatography. Automated Edman degradation and carboxypeptidase Y and B digestion were used to establish the sequence of these peptides. Further fragmentation of two tryptic peptides, T3 and T5, by Staphylococcus aureus protease and cyanogen bromide, respectively, was necessary to complete the sequences. The tryptic peptides accounted for a minimum of 199 residues out of a total of 202 residues predicted by amino acid analysis.

The carboxymethylated a subunit of protocatechuate 3,4-dioxygenase was digested with trypsin. The 14 tryptic peptides were isolated by ion exchange chromatography on DEAE-Sephadex and by gel filtration chromatography.
Automated Edman degradation and carboxypeptidase Y and B digestion were used to establish the sequence of these peptides.
Further fragmentation of two tryptic peptides, T3 and T5, by Staphylococcus aureus protease and cyanogen bromide, respectively, was necessary to complete the sequences. The tryptic peptides accounted for a minimum of 199 residues out of a total of 202 residues predicted by amino acid analysis.

Protocatechuate
3,4-dioxygenase catalyzes the intradiol cleavage of protocatechuic acid by the insertion of 2 atoms of molecular oxygen to form P-carboxy-cis,cis-muconic acid (1, 2). Protocatechuate 3,4-dioxygenase is the second enzyme in the pathway leading fromp-hydroxybenzoate to succinate and acetyl-CoA (3,4). The utilization of this pathway involving two oxygenase reactions enables aerobic bacteria to convert a stable aromatic compound into compounds of central metabolism. It is through this and similar pathways that aromatic biopolymers such as lignin re-enter the carbon cycle.
The native enzyme has a molecular weight of 700,000, contains 8 ferric iron atoms/molecule, and is composed of two types of subunits, a and /?, with molecular weights of 22,500 and 26,800, respectively (5). Electron paramagnetic resonance and Mijssbauer spectroscopy have shown that the environment of the ferric iron in the enzyme is of a previously uncharacterized type, namely, a polar environment with ligands of oxygen or nitrogen atoms, rather than cysteinyl ligands as in other nonheme iron proteins (6). Raman spectroscopy of protocatechuate 3,4-dioxygenase has recently demonstrated that at least one of the iron ligands is a tyrosyl residue (7,8). Extensive spectroscopic studies of protocatechuate 3,4-dioxygenase have provided evidence for an ordered reaction mechanism where binding of the organic substrate is followed by oxygen binding to form a ternary complex. From substrate analogue inhibition data, Mijssbauer spectroscopy, and electron paramagnetic spectroscopy, Que et al. (9) proposed a * This work was supported by Grant PCM 82502 from the National Science Foundation. Partial support was received from the Minnesota Medical Foundation and the University of Minnesota Graduate School to purchase the sequenator. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.&C. Section 1734 solely to indicate this fact.
$ Recipient of National Institutes of Health Predoctoral Trainee Grant I-T32-GM07323.
8 To whom correspondence should be addressed.
mechanism where the 4-hydroxyl group of the substrate is coordinated to the iron. Upon the iron activation of the substrate, the molecular oxygen attacks the aromatic ring to form a peroxy intermediate which rearranges with carboncarbon bond scission (9). Preliminary results with the aldehyde analogue of the substrate indicate that the active site is in the a subunit.' In order to provide a firm basis for further studies on the mechanism of action of protocatechuate 3,4-dioxygenase, the determination of its complete primary structure was undertaken in our laboratory (10). We present in this and the accompanying paper the complete primary sequence of the a subunit. This represents the first primary structure for any dioxygenase and should provide essential information for the eventual understanding of the nature of the interactions between subunits, the structure of the substrate and iron binding sites, and the reaction mechanism. The amino acid sequence is also a necessary prerequisite for the determination of the three-dimensional structure by x-ray crystallography.

MATERIALS AND METHODS AND RESULTS
All materials and procedures used, as well as the "Results," are described in the supplement to this paper.' Fig. 3 and Table II are included in the body of the paper.

DISCUSSION
Fifteen peptides were isolated from the tryptic digest of the a subunit of protocatechuate 3,4-dioxygenase. Their compositions are shown in Table II, and the sequences, which were determined by automated Edman degradation and carboxypeptidase Y digestions, are shown in Fig. 3. Two related peptides, T8A and T8B, were isolated in a ratio of 3:l. These peptides had identical sequences with the exception of a single COOH-terminal arginine in T8A and a diarginyl COOH-terminal sequence in T8B. It was not clear whether TlO, free arginine, arose solely from the partial cleavage of this diarginyl bond or also from other regions of the protein. The composition of the tryptic peptides could account for a minimum of 199 residues out of a total of 202 predicted by amino acid analysis of the whole a subunit.