Primary structures and active site residues of sheep liver serine hydroxymethyltransferase

Abstract

Serine hydroxymethyltransferase (SHMT), which catalyzes the hydroxymethyl transfer from serine to H4-folate, is a unique PLP-enzyme, different from aminotransferases, decarboxylases, racemases, and PLP-enzymes catalyzing - or -elimination and replacement reactions (Figs. 1, 2, and 7). Studies on tryptophan synthase and aspartate aminotransferase (AATase) have provided useful information on the structure and function of PLP-enzymes (Figs. 3–6). Both these enzymes have served as trendsetters in elucidating structure-function relationships among PLP-enzymes.

SHMT, in addition to its special position in the PLP group of enzymes, is part of the “thymidylate cycle” (Fig. 10). It is the first enzyme in the pathway for the interconversion of folate coenzymes (Fig. 9) and plays an important role in the channeling of serine between amino acid and nucleotide metabolism in neoplastic tissues (Fig. 11).

The aim and scope of this study were to determine the primary structure of sheep liver SHMT; compare this structure with other SHMTs and PLP-enzymes; establish evolutionary relationships among SHMTs; determine features common to the primary and secondary structures of PLP-enzymes; identify amino acids essential for enzyme activity; position them in the primary structure; and elucidate the role of these amino acids in the reaction catalyzed by SHMT.

The “Materials and Methods” used in this study, especially the procedure for the large-scale isolation of SHMT (Table 3), manual sequencing by the DABITC/PITC method (Figs. 12 and 13), automated sequencing using a gas-phase sequenator (Figs. 14 and 15), cleavage of SHMT by chemical methods, isolation of peptides by RP-HPLC, electrophoretic separation of peptides, electroblotting, and sequencing of peptides, as well as methods for identification of active site residues, are described in Chapter II.

The purity of the enzyme used in this study was established by native and SDS-PAGE (Figs. 16a, b and 17). The amino acid composition of SHMT was determined by acid hydrolysis (Table 4), indicating the presence of 27 Lys, 27 Arg, 8 Met, and 10 Cys residues in addition to other amino acids. Sheep liver SHMT was pretreated in three different ways: (a) reduction and carboxymethylation, (b) performic acid oxidation, and (c) carboxymethylation using [¹C]-IAA. SHMT modified as described above was digested with trypsin.

The tryptic digest from reduced and carboxymethylated SHMT was separated into groups on a Sephadex G-25 column (Fig. 20). The peptides were further purified by RP-HPLC on an Aquapore RP-300 column (Figs. 21–23). Many peptides were not pure at this stage and were rechromatographed on a Spherisorb 5-ODS column using different gradients. After tryptic digestion, the performic acid–oxidized and [¹C]-IAA labeled SHMT were loaded onto an Aquapore RP-300 column. The separated peptides were repurified on a Spherisorb 5-ODS column (Figs. 25–32). Peptides from the three tryptic digests were sequenced, and the unique sequences were collated (Tables 6–8).

Reduced and carboxymethylated SHMT was digested with chymotrypsin, and the peptides were separated by RP-HPLC using an Aquapore RP-300 column (Fig. 33), followed by a Spherisorb 2-ODS column (Figs. 34–36). Several chymotryptic peptides (Table 9) did not yield new sequences in addition to those obtained by tryptic digestion.

Sheep liver SHMT was subjected to CNBr cleavage (Fig. 45). The peptides were separated by tricine SDS-PAGE (Fig. 38a) and electroblotted onto PVDF membranes (Fig. 38b). The membrane was directly sequenced on a gas-phase sequenator. A representative profile of one cycle of sequencing is shown in Fig. 40. The sequences of the CNBr peptides are listed in Table 10.

To determine the sequence at the N-terminal region, which was blocked by an acetyl group (Fig. 18), cleavage with NHOH was carried out. The N-terminal peptide was isolated, and its amino acid composition determined (Figs. 42, 43 and Table 11).

The complete primary structure of sheep liver SHMT, consisting of 483 amino acids, was determined by aligning the tryptic, chymotryptic, and overlapping CNBr fragments (Fig. 46). The sequence of the sheep liver enzyme was aligned with those of rabbit liver cytosolic and mitochondrial, E. coli, and S. typhimurium enzymes (Fig. 65). The alignment indicated an overall homology of 30% among the various SHMT sequences. Comparison of the sheep liver enzyme sequence with other SHMTs (Table 24) indicated a high degree of homology among the SHMTs sequenced so far.

The secondary structure of sheep liver SHMT, predicted using the Chou and Fasman algorithm (1974a,b), indicated 49% -helical, 14% -strand, and 29% turn content (Fig. 66). Comparison of the hydropathy profiles of the sheep liver enzyme with those of rabbit cytosolic and S. typhimurium enzymes indicated that the plots were similar (Figs. 67 and 68).

A comparison of the primary structure of SHMT with other PLP-enzymes did not reveal any significant homology between sequences (Table 25). However, the secondary structure predictions revealed a common feature—the presence of alternating helices and strands (Table 25, Fig. 69).

Earlier work from this laboratory showed that Arg, His, Cys, and Lys residues were essential for the activity of sheep liver SHMT. The Lys residue involved in forming the internal aldimine with PLP was identified by reducing the enzyme with NaBH (Fig. 47). In this study, the fluorescence of the reduced aldimine was used to locate the PLP-peptide (Fig. 48) instead of the frequently used labeled NaBH for reduction. The peptide was isolated by RP-HPLC (Figs. 49 and 50) and sequenced manually (Fig. 51). The phosphopyridoxyl Lys residue was identified by amino acid analysis following acid hydrolysis (Fig. 52). The sequence of the peptide was compared with the PLP-binding region sequences in other SHMTs (Table 15). The decapeptide V-V-T-T-T-T-H-K-T-L binding PLP was identical in all SHMT sequences, indicating the importance of this region in binding or catalysis. This peptide showed no significant homology with the PLP-peptide in transaminases (Table 16). A comparison of the secondary structure of SHMT and AATase revealed considerable similarity in the PLP-binding region (Fig. 69).

The guanidino group of Arg residues is an excellent candidate for forming hydrogen bonds with the negatively charged carboxyl groups of substrates. PG, a commonly used Arg-modifying reagent, inactivated sheep liver SHMT, and this inactivation was prevented by the presence of H4-folate. Incorporation of [¹C]-PG indicated that two Arg residues were modified per subunit, and this modification was prevented by H4-folate (Table 17).

To locate the sites of PG modification, the enzyme was reacted in the presence and absence of H4-folate using labeled and radioactive PG, respectively. The labeled PG-treated enzyme was digested with trypsin, and the radiolabeled peptides were purified by RP-HPLC (Figs. 53–55). Sequencing of the tryptic peptides indicated that Arg-269 and Arg-462 were the sites of PG modification (Fig. 56, Table 18).

Neither a spectrally discernible quinonoid intermediate (characteristic of the native enzyme when substrates are added, Fig. 57) nor its enhancement by H4-folate was observed with the PG-modified enzyme (Fig. 58). There was no enhancement of the rate of -proton exchange of glycine upon addition of H4-folate, as observed with the native enzyme (Table 19).

Comparison of the sequences of the two Arg residues involved in H4-folate binding—Arg-269 (A-G-M-I-F-Y-R-K) and Arg-462 (A-V-R-A-L-R)—with other SHMT sequences revealed that both residues were conserved in the rabbit cytosolic enzyme, while no local sequence homology existed in prokaryotic enzymes (Table 20).

The Cys residues, Cys-67 and Cys-203, protected by PLP in sheep liver SHMT, were isolated using [¹C]-IAA for carboxymethylation, followed by tryptic digestion of holo- and apo-enzymes (Table 21).

The conformationally buried Cys residues, Cys-247 and Cys-261, were also identified by comparing the radiolabeled peptides obtained from native and denatured holoenzyme (Table 22).

The results describe the primary structure of sheep liver SHMT and its remarkable similarity to rabbit liver cytosolic SHMT. In addition, the observations pinpoint the position of Lys and Arg residues essential for catalysis. The enzyme contains differentially reactive Cys residues, some probably involved in coenzyme binding or facilitating retention of formaldehyde generated at the active site. The sequence around the Lys residue that binds PLP is highly conserved among SHMTs. The Arg residues are involved in H4-folate interaction with the enzyme, which occurs later in the catalytic cycle.