©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Site-directed Mutagenesis of the Subunit of Human Prolyl 4-Hydroxylase
IDENTIFICATION OF THREE HISTIDINE RESIDUES CRITICAL FOR CATALYTIC ACTIVITY (*)

Arja Lamberg , Taina Pihlajaniemi , Kari I. Kivirikko (§)

From the (1) Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, FIN-90220 Oulu, Finland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an tetramer in which the subunits contribute to most parts of the two catalytic sites. To study the roles of histidine and cysteine residues in this catalytic activity we converted all 5 histidines that are conserved between species, 4 nonconserved histidines, and 3 conserved cysteines of the human subunit individually to serine and expressed the mutant subunits together with the wild-type subunit in insect cells by means of baculovirus vectors. Mutation of any of the 3 conserved histidines, residues 412, 483, and 501, inactivated the enzyme completely or essentially completely, with no effect on tetramer assembly or binding of the tetramer to poly(L-proline). These histidines are likely to provide the three ligands needed for the binding of Feto a catalytic site. Mutation of either of the other 2 conserved histidines reduced the amount of enzyme tetramer by 20-25% and the activity of the tetramer by 30-60%. Mutation of the nonconserved histidine 324 totally prevented tetramer assembly, whereas mutation of the 3 other nonconserved histidines had no effects. Two of the 3 cysteine to serine mutations, those involving residues 486 and 511, totally prevented tetramer assembly under the present conditions, whereas the third, involving residue 150, had only a minor effect in reducing tetramer assembly and activity. The data do not support previous suggestions that cysteine residues are involved in Febinding sites. Additional mutagenesis experiments demonstrated that the two glycosylated asparagines have no role in tetramer assembly or catalytic activity.


INTRODUCTION

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens and related proteins by the hydroxylation of proline residues in - X-Pro-Gly- sequences. This modification plays a central role in the synthesis of all collagens, as 4-hydroxyproline residues are essential for the folding of the newly synthesized collagen polypeptide chains into triple-helical molecules (for reviews, see Refs. 1-3).

Prolyl 4-hydroxylase requires Fe, 2-oxoglutarate, O, and ascorbate. The active enzyme from vertebrates is an tetramer in which the subunits contribute to most parts of the two catalytic sites (1, 2, 3) . A catalytic site appears to comprise a set of separate locations for binding of the peptide substrate and the various cosubstrates. The Feis probably coordinated with the enzyme by three side chains (1, 2, 3, 4) . The 2-oxoglutarate binding site can be divided into distinct subsites; subsite I is probably a positively charged side chain of the enzyme that ionically binds the C-5 carboxyl group of 2-oxoglutarate, whereas subsite II consists of two cis-positioned coordination sites for the enzyme-bound Fewhich is chelated by the C-2 oxo and C-1 carboxyl functions (1, 2, 3, 4, 5) . Binding of Oand decarboxylation of 2-oxoglutarate is assumed to lead to the formation of ferryl ion, which subsequently hydroxylates a proline residue (1, 3, 4) . Ascorbate acts as an alternative oxygen acceptor in the uncoupled decarboxylation cycles, in which the 2-oxoglutarate is decarboxylated without subsequent hydroxylation of the peptide substrate (6, 7) . The ascorbate binding site also contains the two cis-positioned coordination sites for the enzyme-bound iron and is thus partially identical to the binding site of 2-oxoglutarate (8) .

Complete cDNA-derived amino acid sequences have now been reported for the subunit of prolyl 4-hydroxylase from man (9) , chick (10) , and Caenorhabditis elegans (11) and for the subunit from several species (2, 12, 13, 14, 15, 16) . Surprisingly, the subunit was found to be identical to the protein disulfide-isomerase (EC 5.3.4.1) and to have protein disulfide-isomerase activity even when present in the prolyl 4-hydroxylase tetramer (12, 13, 17) . This protein disulfide-isomerase/ subunit has subsequently been found to be a highly unusual multifunctional polypeptide (2, 3, 15, 16) .

Experiments on the inactivation of prolyl 4-hydroxylase by diethyl pyrocarbonate and the prevention of this inactivation by cosubstrates of the reaction have suggested that histidine residues are functional at the catalytic sites of the enzyme, probably at the Febinding sites (18) . A search for conserved amino acids within the sequences of several 2-oxoglutarate dioxygenases demonstrated a weak homology within 2-histidine-containing motifs located about 50-70 amino acids apart, the histidines concerned being residues 412 and 483 in the human subunit sequence (18) .

In order to elucidate the roles of histidine residues of the subunit in the catalytic activity of prolyl 4-hydroxylase, we converted all 5 histidines that are conserved between species from man to C. elegans and 4 additional nonconserved histidines individually to serine and expressed the mutant human subunits together with the wild-type protein disulfide-isomerase/ subunit in insect cells (19, 20) by means of baculovirus vectors.

It has also been suggested that cysteine residues may be functional at the Febinding sites of prolyl 4-hydroxylase (3) . Two cysteine residues of the subunit have been reported to form an intrachain disulfide bond that is essential for tetramer assembly (21) . We studied here the possible roles of the 3 other cysteine residues in catalytic activity. In addition, the roles of the 2 glycosylated asparagine residues were studied.


MATERIALS AND METHODS

Site-directed Mutagenesis

Histidines 63, 141, 165, 221, 296, 324, 412, 483, and 501 (codon CAC or CAT) and cysteines 150, 486, and 511 (codon TGC or TGT) in the human subunit were converted individually to serine (codon TCC). The 2 asparagines, residues 96 (codon AAC) and 242 (codon AAT), which serve as the N-glycosylation sites, were converted to glutamines (codon CAG) in a double mutant subunit. The site-directed mutagenesis steps were performed in a pBluescript (Stratagene) vector containing the full-length cDNA clone (PA-59) of the human subunit at the SmaI site (9) . The mutagenesis was carried out using an oligonucleotide-directed in vitro mutagenesis system based on the unique site elimination procedure (Pharmacia Biotech Inc.), after which the plasmid was digested with PstI and BamHI, the cleavage sites for which closely flank the SmaI site. The resulting PstI- PstI and PstI and BamHI fragments containing 61 bp of the 5`-untranslated sequence, the whole coding region, and 551 bp of the 3`-untranslated sequence were then cloned into the PstI- BamHI site of the baculovirus transfer vector pVL1392 (22) . The sequences were verified by Sanger dideoxynucleotide sequencing (23) .

Generation of Recombinant Baculoviruses

The recombinant baculovirus transfer vectors were cotransfected into Spodoptera frugiperda Sf9 insect cells with a modified Autographa californica nuclear polyhedrosis virus DNA (PharMingen) by calcium phosphate transfection, and the recombinant viruses were selected (24) . The resultant viruses encoding the mutant subunit sequences were termed -H63S, -H141S, -H165S, -H221S, -H296S, -H324S, -H412S, -H483S, -H501S, -C150S, -C486S, -C511S, and -N96Q,N242Q.

Analysis of Recombinant Proteins in Insect Cells

The insect cells (Sf9 or High Five, Invitrogen) were cultured in TNM-FH medium (Sigma) supplemented with 10% fetal bovine serum (BioClear) at 27 °C. The cells seeded at a density 10/ml were infected at a multiplicity of 5 with any of the viruses coding for the mutant subunit together with a virus coding for the protein disulfide-isomerase/ subunit (19) . The cells were harvested 72 h after infection, washed twice with a solution of 0.15 M NaCl and 0.02 M phosphate, pH 7.4, homogenized in a 0.1 M NaCl, 0.1 M glycine, 10 µM dithiothreitol, 0.1% Triton X-100, and 0.01 M Tris buffer, pH 7.8, and centrifuged at 10,000 g for 20 min. Aliquots of the supernatants were analyzed by denaturing 8% SDS-PAGE() or nondenaturing 8% PAGE and assayed for enzyme activity.

Binding of Mutant Enzymes to Poly(L-proline)

Binding of prolyl 4-hydroxylase tetramers containing some of the mutant subunits to poly(L-proline) was studied by mixing 0.5-ml aliquots of the supernatants of cell homogenates expressing the wild-type enzyme or a mutant enzyme tetramer with 0.5 ml of a suspension containing poly(L-proline) coupled to agarose (25) . After gentle stirring for 6 h at 4 °C, the poly(L-proline)-agarose was separated out by centrifugation at 10,000 g for 5 min, and the resulting supernatants were analyzed by nondenaturing 8% PAGE and Coomassie Brilliant Blue staining.

Purification of Prolyl 4-Hydroxylase

The prolyl 4-hydroxylase tetramer containing the double mutant N96Q,N242Q subunit was purified by a procedure consisting of poly(L-proline) affinity chromatography and DEAE-cellulose chromatography (26) .

Other Assays

Prolyl 4-hydroxylase activity was assayed by a method based on the hydroxylation-coupled decarboxylation of 2-oxo[1-C]glutarate (25) . Kvalues were determined as described previously (27) . Protein concentrations were determined with a Bio-Rad protein assay kit (Bio-Rad). The levels of expression of the wild-type and mutant prolyl 4-hydroxylase tetramers were compared by densitometry of the Coomassie Brilliant Blue-stained bands in nondenaturing PAGE using a bioimage (BioImage, Millipore).


RESULTS

Selection of Residues for Mutagenesis

The subunit of human prolyl 4-hydroxylase has 12 histidine residues (Fig. 1). One of them is located in the alternatively spliced region in which the sequence coded by exon 9 contains a histidine in position 349, whereas the sequence coded by exon 10 contains it in position 360 (28) . A recently cloned prolyl 4-hydroxylase subunit isoform, termed the (II) subunit, has been found to form an ((II))tetramer with the protein disulfide-isomerase/ subunit (29) . The Kvalues of this tetramer for the cosubstrates and the peptide substrate are very similar to those of the tetramer studied here, but the sequence of the (II) subunit contains an arginine in the position corresponding to histidine 349 (29) . Thus histidine 349/360 can be regarded as a nonconserved residue. Of the remaining 11 histidines, only 5 ( circled in Fig. 1) are conserved between the human and C. elegans subunits (9, 11) . All these, together with 4 nonconserved histidines (these 9 histidines are underlined in Fig. 1) were converted individually to serine.


Figure 1: Schematic representation of the subunit of human prolyl 4-hydroxylase. Numbering of the amino acids begins with the first residue in the processed subunit, the signal sequence being indicated by a shaded box and the alternatively spliced region by a black box. All histidine and cysteine residues and the 2 glycosylated asparagine residues are shown. Conserved residues (see text) are circled, and residues subjected to mutation in the present experiments are underlined. This numbering differs from that used initially (9) in that the initial numbering began with the first residue of the signal peptide.



All 5 cysteine residues (Fig. 1) are conserved in the subunit from man to C. elegans (9, 10, 11) . Recent work on assembly of the prolyl 4-hydroxylase tetramer in a cell-free system (30) has demonstrated that cysteines 276 and 293 form an intrachain disulfide bond that is essential for tetramer assembly (21) . Cysteines 486 and 511 may form another intrachain disulfide, but they are not essential for tetramer assembly. Cysteine 150 does not seem to be involved in any disulfide bonding (21) . The possible catalytic roles of cysteines as 150, 486, and 511 were studied here by converting these individually to serine.

The human subunit contains 2 asparagine residues that act as attachment sites for oligosaccharide units (asparagines 96 and 242, Fig. 1). These vary greatly in the extent of their glycosylation, the polypeptide being present in diglycosylated, monoglycosylated, and nonglycosylated forms (19, 31) . Both these asparagines were converted to glutamines in a double mutant subunit.

Histidine to Serine Mutants

In order to study whether any of the 9 histidine mutations inhibits assembly of the prolyl 4-hydroxylase tetramer, each of the mutant subunits was expressed in insect cells together with the wild-type protein disulfide-isomerase/ subunit. The cells were harvested 72 h after baculovirus infection, homogenized in a buffer containing 0.1% Triton X-100, and centrifuged. The Triton X-100-soluble proteins were then analyzed by PAGE performed under nondenaturing conditions and Coomassie staining (Fig. 2). A distinct band corresponding to the enzyme tetramer (11, 19, 20) was seen in the Triton X-100-soluble fraction from cells infected with a virus coding for either the wild-type subunit or any of 8 out of the 9 histidine to serine mutant subunits. The only mutant subunit that did not form a tetramer was that containing the histidine to serine mutation in position 324. Surprisingly, this is one of the 4 nonconserved histidines studied here, this position being occupied by lysine in the C. elegans subunit (11) . Repeated control experiments indicated that this result was not due to a methodological artifact. As the goal of this work was to identify residues critical for catalytic activity rather than tetramer assembly, this aspect was not pursued any further.


Figure 2: Nondenaturing PAGE analysis of prolyl 4-hydroxylase tetramer formation from wild-type or histidine to serine mutant human subunits and the wild-type human protein disulfide-isomerase/ subunit expressed in insect cells by means of baculovirus vectors. The samples were extracted with a buffer containing 0.1% Triton X-100 and analyzed by nondenaturing PAGE and Coomassie staining. Lanes 1 and 12, extracts from cells infected with the wild-type subunit-coding virus and wild-type protein disulfide-isomerase/ subunit-coding virus; lanes 2-10, extracts from cells infected with a mutant subunit-coding virus and the wild-type protein disulfide-isomerase/ subunit-coding virus, the subunits having the following mutations: H63S ( lane 2), H141S ( lane 3), H165S ( lane 4), H221S ( lane 5), H296S ( lane 6), H324S ( lane 7), H412S ( lane 8), H483S ( lane 9), and H501S ( lane 10). Lane 11, extract from cells infected with the protein disulfide-isomerase/ subunit-coding virus alone. The samples in lanes 1-6 and 10-12 were run in the same experiment and those in lanes 7-9 in a second experiment. The arrows indicate the enzyme tetramer () and the nonassociated protein disulfide-isomerase/ subunit (). The nonassociated subunit is insoluble in 0.1% Triton X-100 and is not seen.



Densitometric tracings of the Coomassie-stained bands corresponding to the enzyme tetramer from at least four experiments in the case of each mutant indicated that there are no major differences in the amounts of tetramer formed per unit extractable cell protein except in the case of the histidine 324 mutation described above. However, two of the mutant subunit-coding viruses, those with histidine 165 or 221 mutation, consistently produced about 20-25% less tetramer than the other viruses (details not shown).

To study the effect of the various histidine mutations on the prolyl 4-hydroxylase activity of the tetramer, Triton X-100 extracts from cell homogenates were assayed by a method based on measurement of the hydroxylation-coupled decarboxylation of 2-oxo[1-C]glutarate (25) . All values were expressed per 10 µg of extractable cell protein ().

Three histidine to serine mutations were found to have a marked effect on prolyl 4-hydroxylase activity in the resulting tetramer (). A mutation in position 412 or 483 totally eliminated the enzyme activity, whereas a mutation in position 501 reduced it to about 4% (). None of the mutations involving the 3 nonconserved histidines that did not inhibit tetramer assembly, residues 63, 141, and 296, had any effect on enzyme activity of the tetramer. The two mutations of conserved histidines that appeared to have a minor inhibitory effect on the amount of enzyme tetramer, residue 165 and 221 (see above), produced a reduction of about 70 or 50%, respectively, in enzyme activity per unit extractable cell protein relative to that obtained with the wild-type subunit (). Since the amount of tetramer was reduced by about 20-25%, however (see above), the actual reduction in the activity of the tetramer was about 60 or 30%. The Kvalues for Fe, 2-oxoglutarate, ascorbate, and the peptide substrate with these two mutant tetramers were identical to those with the wild-type enzyme (details not shown).

Further investigations into the properties of the histidine 501 to serine mutant enzyme showed no differences in the Kvalues for Fe, ascorbate, or the peptide substrate relative to the wild-type enzyme, but the Kfor 2-oxoglutarate was consistently about 3-fold (). In agreement with this minor difference, the Kfor pyridine-2,4-dicarboxylate, which acts as a competitive inhibitor with respect to 2-oxoglutarate, was about 2.5-fold (). The main difference, however, was that the Vdetermined from the kinetic plots was consistently less than about 5% of that of the wild-type enzyme (details not shown).

Because prolyl 4-hydroxylase becomes bound to poly(L-proline) even in the absence of Feand other cosubstrates (3, 25, 32) , it seemed possible to determine whether the three histidine mutations that had a marked effect on enzyme activity had any effect on binding to poly(L-proline). Small aliquots of Triton X-100 extracts from cells expressing either the wild-type enzyme or an enzyme containing any of the three mutant subunits with a histidine 412, 483, or 501 to serine substitution were mixed with poly(L-proline) linked to agarose. The recently discovered ((II))tetramer (see above), which differs from the tetramer studied here in that it does not bind to poly(L-proline) (29) , served as a control. All three mutant enzymes became bound to poly(L-proline) as efficiently as the wild-type enzyme, whereas no binding was detected with the ((II))tetramer (Fig. 3). These data indicate that none of the three histidine mutations with a marked effect on enzyme activity had any effect on the binding of the enzyme to poly(L-proline).


Figure 3: Nondenaturing PAGE analysis of the binding of prolyl 4-hydroxylase tetramers containing certain wild-type or histidine to serine mutant subunits to poly(L-proline) linked to agarose. Small aliquots of Triton X-100 extracts from cells expressing either a wild-type enzyme tetramer or a mutant tetramer were mixed with poly(L-proline) linked to agarose. After gentle stirring for 6 h at 4 °C, the poly(L-proline)-agarose was separated out by centrifugation, and the resulting supernatants were analyzed by nondenaturing PAGE and Coomassie staining. A, samples analyzed before incubation with poly(L-proline)-agarose; B, samples analyzed after incubation. Lanes 1, extract from cells infected with the wild-type subunit-coding virus and wild-type protein disulfide-isomerase/ subunit-coding virus; lanes 2, extract from cells infected with the mouse type II subunit (see text) and human protein disulfide-isomerase/ subunit-coding viruses; lanes 3-5, extracts from cells infected with a mutant subunit and wild-type protein disulfide-isomerase/ subunit-coding viruses, the subunits having the following mutations: H412S ( lane 3), H483S ( lane 4), H501S ( lane 5). The arrows indicate the enzyme tetramer () and the nonassociated protein disulfide-isomerase/ subunit ().



Cysteine to Serine Mutants

As in the case of the histidine mutants, each of the cysteine mutant subunits was expressed in insect cells together with the wild-type protein disulfide-isomerase/ subunit, and the Triton X-100-soluble proteins were used for the analyses. Two out of the three cysteine mutants, those involving residues 486 and 511, were found either to prevent tetramer assembly or to destabilize thetetramer to such an extent that no tetramer band was seen by PAGE performed under nondenaturing conditions and Coomassie staining (Fig. 4). The third cysteine mutant, that involving residue 150, had no major effect on tetramer assembly (Fig. 4), but densitometric tracings of the Coomassie-stained band indicated that the amount of tetramer formed was slightly reduced, being about 80% of that seen with most of the other viruses (details not shown). The amount of prolyl 4-hydroxylase activity observed with the cysteine 150 mutant subunit had decreased to a slightly larger extent than the amount of tetramer (I), suggesting that the specific activity of the mutant tetramer was about 80% of that of the wild-type enzyme. The two other cysteine mutants that appeared to prevent tetramer assembly generated no prolyl 4-hydroxylase activity (I).


Figure 4: Nondenaturing PAGE analysis of prolyl 4-hydroxylase tetramer formation from wild-type or mutant human subunits and wild-type human protein disulfide-isomerase/ subunit expressed in insect cells by means of baculovirus vectors. The samples were extracted with a buffer containing 0.1% Triton X-100 and analyzed by nondenaturing PAGE and Coomassie staining. Lanes 1 and 7, extracts from cells infected with the wild-type subunit-coding virus and wild-type protein disulfide-isomerase/ subunit-coding virus; lanes 2-5, extracts from cells infected with a mutant subunit-coding virus and the wild-type protein disulfide-isomerase/ subunit-coding virus, the subunits having the following mutations: N96Q,N242Q double mutation ( lane 2), C150S ( lane 3), C486S ( lane 4), and C511S ( lane 5). Lane 6, extract from cells infected with the protein disulfide-isomerase/ subunit-coding virus alone. The arrows indicate the enzyme tetramer () and the nonassociated protein disulfide-isomerase/ subunit ().



Asparagines 96 and 242 to Glutamine Double Mutant

An subunit containing both these mutations was found to form a tetramer, as shown by analysis of the Triton X-100-soluble proteins by PAGE performed under nondenaturing conditions (Fig. 4). Densitometric tracings of the Coomassie-stained band indicated no decrease in the amount of tetramer formed. The amount of prolyl 4-hydroxylase activity observed with the double mutant subunit was identical to that of the wild-type enzyme (I). When the tetramer was purified by an affinity column procedure and analyzed by SDS-PAGE under reducing conditions, the size of the double mutant subunit was distinctly smaller than that of either the diglycosylated or monoglycosylated subunit present in the wild-type enzyme, the difference being consistent with loss of all the carbohydrate (Fig. 5).


Figure 5: SDS-PAGE analysis under reducing conditions of purified prolyl 4-hydroxylase tetramers containing either the wild-type or asparagine to glutamine double mutant subunit. The wild-type enzyme is shown in lane 2 and the mutant enzyme in lane 3. Molecular weight markers were run in lane 1. The gel was stained with Coomassie Brilliant Blue. The arrows indicate the diglycosylated and monoglycosylated wild-type subunits ( -wt), the nonglycosylated asparagines 96 and 242 to glutamine double mutant subunit ( -N96Q,N242Q), and the wild-type protein disulfide-isomerase/ subunit ().




DISCUSSION

The present data demonstrate that there are 3 conserved histidine residues that play a major role in the catalytic activity of prolyl 4-hydroxylase. Mutation of either histidine 412 or 483 to serine completely inactivated the enzyme, whereas mutation of histidine 501 to serine reduced its activity to about 5%. These results agree with previous data indicating that the enzyme is inactivated by diethyl pyrocarbonate, an effect which could be partially prevented by the presence of Feand 2-oxoglutarate or Feand ascorbate but not by the peptide substrate, even though the peptide becomes bound to the enzyme in the absence of Feand other cosubstrates (18) . Protection by Fealone could not be studied, as Fewill not remain bound to the enzyme under nonturnover conditions (3, 18) .

The Feis probably coordinated with a catalytic site of prolyl 4-hydroxylase by means of three side chains (1, 2, 3, 4) . Some early results suggested that one or more of these side chains may be cysteine residues (3) . Nevertheless, the present data are not consistent with this suggestion, as mutation of the only cysteine that was not essential for tetramer assembly or stability, residue 150, had practically no effect on the catalytic activity of the tetramer. Analyses of another 2-oxoglutarate dioxygenase, isopenicillin N synthase by a variety techniques, suggest that all three Febinding ligands are histidines (33, 34, 35) .

The most likely role for all three critical histidines identified here is that they provide the three ligands involved in the coordination of Fewith a catalytic site. As the main binding site for both 2-oxoglutarate (4, 5) and ascorbate (8) consists of the enzyme-bound iron atom, it is unlikely that mutation of a residue contributing to an additional subsite of either a 2-oxoglutarate or ascorbate binding site would lead to such a marked inactivation of the enzyme. Although mutation of histidine 501 increased the Kfor 2-oxoglutarate and the Kfor pyridine-2,4-dicarboxylate about 3-fold, this increase is not consistent with the possibility that histidine 501 may provide the positively charged side chain that interacts with the negatively charged carboxyl group at C-5 of 2-oxoglutarate and is termed subsite I (5) . The Kof the wild-type enzyme for pyridine-2,4-dicarboyxylate, which is able to bind at subsites I and II (subsite II being Fe), is less than 1/10 of that of pyridine-2-carboxylate, which lacks a domain able to bind at subsite I (5) . Accordingly, mutation of the positively charged subsite I residue is likely to lead to a much more than a 3-fold increase in the Kfor 2-oxoglutarate and the Kfor pyridine-2,4-dicarboxylate.

The finding that the histidine 412, 483, and 501 mutant enzymes all became bound to poly(L-proline) clearly demonstrates that none of these histidines is critically involved in a poly(L-proline) binding site. This finding also suggests that none of these mutations caused any marked overall changes in the structure of the enzyme, as the sites involved in poly(L-proline) binding appear to have retained their native-type conformations. The data indicating that none of these three histidine mutations inhibited assembly of the prolyl 4-hydroxylase tetramer likewise suggest that these mutations have caused no major structural changes.

A histidine-containing motif showing homology to that around histidine 483 in the subunit of human prolyl 4-hydroxylase has recently been identified in bovine aspartyl (asparaginyl) -hydroxylase (36) . When the histidine present in this motif was converted to alanine, no enzyme activity was detected in the resulting mutant enzyme (36) . Also, the mutant enzyme failed to bind Fe/2-oxoglutarate, suggesting that the histidine residue is involved in the binding of either Feor 2-oxoglutarate. The data further suggested that no major structural changes had taken place as a result of the mutation (36) , the results being in a complete agreement with those obtained here with prolyl 4-hydroxylase. Sequence analyses of flavone 3 hydroxylase from several sources also demonstrated the presence of three conserved histidines which may be involved in the binding of Feto the catalytic site (37) .

Three histidine to serine mutations appeared to influence the tetramer assembly. Surprisingly, mutation of one of the 4 nonconserved histidines, residue 324, completely prevented tetramer formation, indicating that although this position can be occupied by either histidine or lysine (11) , its occupation by serine will prevent tetramer assembly. Mutation of two conserved histidines, residue 165 or 221, had more minor effects on tetramer assembly or stability. The amount of enzyme activity obtained with these two mutants decreased to an even larger extent than the amount of tetramer, suggesting that the mutations caused some minor structural changes. The cysteine 150 to serine mutation may also have caused minor structural changes, as the amount of tetramer was consistently slightly lower, whereas the two other cysteine mutations studied, those involving residues 486 and 511, totally eliminated tetramer assembly. This result differs from those obtained in experiments on assembly of the prolyl 4-hydroxylase tetramer in a cell-free system, in that mutation of either cysteine 486 or 511 reduced the amount of tetramer formed but did not totally prevent tetramer assembly (21) . The data obtained both here and in a cell-free system (21) suggest that these 2 cysteines play a major role in maintaining the native-type structure of the subunit, probably by intrachain disulfide bond formation, and thus these 2 cysteines are not likely to be directly involved in the catalytic mechanism of the enzyme.

The functions of the asparagine-linked carbohydrate units present in the subunit of prolyl 4-hydroxylase are unknown (3) , and it has been demonstrated that enzymic removal of the carbohydrate from the tetramer has little if any effect on the enzyme activity (31) . The present data demonstrate that mutation of both glycosylated asparagines to glutamines causes no changes in tetramer assembly or in the specific activity of the resulting tetramer. As the extent of glycosylation of these 2 asparagines in the wild-type prolyl 4-hydroxylase is highly heterogeneous, the carbohydrate-free tetramer produced here may be more suitable than the wild-type tetramer for attempts to crystallize the enzyme.

  
Table: Prolyl 4-hydroxylase activity of Triton X-100 extracts from cells expressing various histidine to serine mutant subunits together with the wild-type protein disulfide-isomerase/ subunit


  
Table: Kvalues for cosubstrates and the peptide substrate and Kvalue for pyridine-2,4-dicarboxylate of the enzyme tetramer containing the histidine 501 to serine mutant subunit

All values have been determined in Triton X-100 extract from cells expressing either the wild-type or mutant tetramer as the source of the enzyme. The Kobtained for 2-oxoglutarate with this crude wild-type enzyme is about twice that obtained with the pure enzyme (19).


  
Table: Prolyl 4-hydroxylase activity of Triton X-100 extracts from cells expressing various cysteine to serine mutant subunits or the asparagine to glutamine double mutant subunit together with the wild-type protein disulfide-isomerase/ subunit

For polypeptide nomenclature and measurement units, see Table I.



FOOTNOTES

*
This work was supported by grants from the Medical Research Council of the Academy of Finland. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Medical Biochemistry, University of Oulu, Kajaanintie 52A, FIN-90220, Oulu, Finland. Tel.: 358-81-5375801; Fax: 358-81-5375810.

The abbreviation used is: PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Eeva Lehtimäki and Riitta Polojärvi for their expert technical assistance.


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