Involvement of aspartic and glutamic residues in kringle-2 of tissue-type plasminogen activator in lysine binding, fibrin binding and stimulation of activity as revealed by chemical modification and oligonucleotide-directed mutagenesis

Modification of glutamic and aspartic acid residues of tissue-type plasminogen activator (t-PA) with 1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimide leads to a decrease in affinity for lysine and fibrin, to a decrease of plasminogen activation activity in the presence of a fibrin mimic, but leaves amidolytic activity and plasminogen activation without fibrin mimic unaffected. Experiments with kringle-2 ligands and a deletion mutant of t-PA (K2P) suggests that glutamic or aspartic acid residues in K2 of t-PA are involved in stimulation of activity, lysine binding and fibrin binding. Mutant t-PA molecules were constructed by site-directed mutagenesis in which one or two of the five aspartic or glutamic acid residues in K2 were changed to asparagine or glutamine respectively. Mutation of Asp236 and/or Asp238 leads to t-PA molecules with 3- to 4-fold lower specific activity in the presence of fibrin mimic and having no detectable affinity for lysine analogs. However, fibrin binding was not influenced. Mutation of Glu254 also leads to a 3- to 4-fold lower activity, but to a much smaller reduction of lysine or fibrin binding. Residues Asp236 and Asp238 are both essential for binding to lysine derivatives, while Glu254 might be involved but is not essential. Residues Asp236, Asp238 and Glu254 are all three involved in stimulation of activity. Remarkably, mutation of residues Asp236 and/or Asp238 appears not to influence fibrin binding of t-PA whereas that of Glu254 does.     

Site-directed mutagenesis of catalytic active-site residues of Taka-amylase A.

The cDNA encoding Taka-amylase A (EC.3.2.1.1, TAA) was isolated to identify functional amino acid residues of TAA by protein engineering. The putative catalytic active-site residues and the substrate binding residue of TAA were altered by site-directed mutagenesis: aspartic acid-206, glutamic acid-230, aspartic acid-297, and lysine-209 were replaced with asparagine or glutamic acid, glutamine or aspartic acid, asparagine or glutamic acid, and phenylalanine or arginine, respectively. Saccharomyces cerevisiae strain YPH 250 was transformed with the expression plasmids containing the altered cDNA of the TAA gene. All the transformants with an expression vector containing the altered cDNA produced mutant TAAs that cross-reacted with the TAA antibody. The mutant TAA with alteration of Asp206, Glu230, or Asp297 in the putative catalytic site had no alpha-amylase activity, while that with alteration of Lys209 in the putative binding site to Arg or Phe had reduced activity.     

Purification, characterization and gene cloning of a novel glutamic acid-specific endopeptidase from Staphylococcus aureus ATCC 12600.

Twenty strains of Staphylococcus aureus from ATCC type cultures and strains found in clinical studies were cultivated, and their endopeptidase activity specific for glutamic acid was surveyed using benzyloxycarbonyl-Phe-Leu-Glu-p-nitroanilide (Z-Phe-Leu-Glu-pNA) as a substrate. The activity was found in two of the strains, ATCC 12600 and ATCC 25923. A glutamic acid-specific proteinase, which we propose to call SPase, was purified from the culture filtrate of S. aureus strain ATCC 12600 by a series of column chromatographies on DEAE-Sepharose twice and on Sephacryl S-200. A single band was observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified SPase. The molecular weight of the proteinase was estimated to be 34000 by SDS-PAGE. When synthetic peptides and oxidized insulin B-chain were used as substrates, SPase showed the same substrate specificity as V8 proteinase, EC 3.4.21.9, which specifically cleaves peptide bonds on the C-terminal side of glutamic acid and aspartic acid. Examination with p-nitroanilides of glutamic acid and aspartic acid as substrates, however, revealed that both proteinases are highly specific for a glutamyl bond in comparison with an aspartyl bond. To elucidate the complete primary structure of SPase, its gene was cloned from genomic DNA of S. aureus ATCC 12600, and the nucleotide sequence was determined. Taking the amino acid sequence of SPase from the NH2-terminus to the 27th residue into consideration, the clones encode a mature peptide of 289 amino acids, which follows a prepropeptide of 68 residues. SPase was confirmed to be a novel endopeptidase specific for glutamic acid, being different from V8 proteinase which consists of 268 amino acids.     

Aspartic acid-66 is the only essential negatively charged residue in the putative hydrophilic loop region of the metal-tetracycline/H+ antiporter encoded by transposon Tn10 of Escherichia coli.

Of the 16 acidic amino acid residues located in the hydrophilic region of the metal-tetracycline/H+ antiporter of transposon Tn10, five glutamic acids and three aspartic acids are conserved among the tetracycline/H+ antiporters of Gram-negative bacteria. When these conserved acidic residues were each replaced by a neutral polar residue, glutamine or asparagine, only the Asp66 substitution mutants completely lost their transport activity. The substitution of Glu274, Asp120, Glu181, or Asp38 caused significant reduction of the transport activity, whereas the substitution of the other three residues had no detectable effect on the activity. These findings led to the conclusion that only Asp66 is essential for the transport function.     

Structure and possible catalytic residues of Taka-amylase A

A complete molecular model of Taka-amylase A consisting of 478 amino acid residues was built with the aid of amino acid sequence data. Some typical structural features of the molecule are described. A model fitting of an amylose chain in the catalytic site of the enzyme showed a possible productive binding mode between substrate and enzyme. On the basis of the difference Fourier analysis and the model fitting study, glutamic acid (Glu230) and aspartic acid (Asp297), which are located at the bottom of the cleft, were concluded to be the catalytic residues, serving as the general acid and base, respectively.     

Functional consequences of single amino acid substitutions in calmodulin-activated adenylate cyclase of Bordetella pertussis.

Calmodulin-activated adenylate cyclase of Bordetella pertussis and Bacillus anthracis are two cognate bacterial toxins. Three short regions of 13-24 amino acid residues in these proteins exhibit between 66 and 80% identity. Site-directed mutagenesis of four residues in B. pertussis adenylate cyclase situated in the second (Asp188, Asp190) and third (His298, Glu301) segments of identity were accompanied by important decrease, or total loss, of enzyme activity. The calmodulin-binding properties of mutated proteins showed no important differences when compared to the wild-type enzyme. Apart from the loss of enzymatic activity, the most important change accompanying replacement of Asp188 by other amino acids was a dramatic decrease in binding of 3'-anthraniloyl-2'-deoxyadenosine 5'-triphosphate, a fluorescent analogue of ATP. From these results we concluded that the two neighbouring aspartic acid residues in B. pertussis adenylate cyclase, conserved in many other ATP-utilizing enzymes, are essential for binding the Mg(2+)-nucleotide complex, and for subsequent catalysis. Replacement of His298 and Glu301 by other amino acid residues affected the nucleotide-binding properties of adenylate cyclase to a lesser degree suggesting that they might be important in the mechanism of enzyme activation by calmodulin, rather than being involved directly in catalysis.     

Substitution of membrane-embedded aspartic acids in bacteriorhodopsin causes specific changes in different steps of the photochemical cycle

Millisecond photocycle kinetics were measured at room temperature for 13 site-specific bacteriorhodopsin mutants in which single aspartic acid residues were replaced by asparagine, glutamic acid, or alanine. Replacement of aspartic acid residues expected to be within the membrane-embedded region of the protein (Asp-85, -96, -115, or -212) produced large alterations in the photocycle. Substitution of Asp-85 or Asp-212 by Asn altered or blocked formation of the M410 photointermediate. Substitution of these two residues by Glu decreased the amount of M410 formed. Substitutions of Asp-96 slowed the decay rate of the M410 photointermediate, and substitutions of Asp-115 slowed the decay rate of the O640 photointermediate. Corresponding substitutions of aspartic acid residues expected to be in cytoplasmic loop regions of the protein (Asp-36, -38, -102, or -104) resulted in little or no alteration of the photocycle. Our results indicate that the defects in proton pumping which we have previously observed upon substitution of Asp-85, Asp-96, Asp-115, and Asp-212 [Mogi, T., Stern, L. J., Marti, T., Chao, B. H., & Khorana, H. G. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 4148-4152] are closely coupled to alterations in the photocycle. The photocycle alterations observed in these mutants are discussed in relation to the functional roles of specific aspartic acid residues at different stages of the bacteriorhodopsin photocycle and the proton pumping mechanism.     

Structure-function studies of the non-heme iron active site of isopenicillin N synthase: some implications for catalysis

Isopenicillin N synthase (IPNS) is a non-heme ferrous iron-dependent oxygenase that catalyzes the ring closure of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to form isopenicillin N. Spectroscopic studies and the crystal structure of IPNS show that the iron atom in the active species is coordinated to two histidine and one aspartic acid residues, and to ACV, dioxygen and H2O. We previously showed by site-directed mutagenesis that residues His212, Asp214 and His268 in the IPNS of Streptomyces jumonjinensis are essential for activity and correspond to the iron ligands identified by crystallography. To evaluate the importance of the nature of the protein ligands for activity, His214 and His268 were exchanged with asparagine, aspartic acid and glutamine, and Asp214 replaced with glutamic acid, histidine and cysteine, each of which has the potential to bind iron. Only the Asp214Glu mutant retained activity, approximately 1% that of the wild type. To determine the importance of the spatial arrangement of the protein ligands for activity, His212 and His268 were separately exchanged with Asp214; both mutant enzymes were completely defective. These findings establish that IPNS activity depends critically on the presence of two histidine and one carboxylate ligands in a unique spatial arrangement within the active site. Molecular modeling studies of the active site employing the S. jumonjinensis IPNS crystal structure support this view. Measurements of iron binding by the wild type and the Asp214Glu, Asp214His and Asp214Cys-modified proteins suggest that Asp214 may have a role in catalysis as well as in iron coordination.     

Analysis of the conformations corresponding to hexapeptide and large sequences characterized by continuous single amino acid repeats in proteins

The analysis of conformations corresponding to continuous amino acid repeat peptides (CARPs) comprising six or more residues in proteins of known three-dimensional structure revealed that alanine, glycine, glutamic acid, proline, valine, histidine, aspartic acid, glutamine and lysine were associated as repeating amino acid residues. Alanine, glycine and histidine CARPs were most common, although the histidine hexapeptide and large CARPs mainly correspond to affinity tags and are not part of the native protein sequence. The Ala and Glu CARPs were observed either as part of helix, or coil or a combination of these conformations. The octapeptide Ala CARP in six-hairpin glycosidases was observed as part of strand and coil conformation. The Gly and Pro CARPs were mainly associated with coil conformation. Majority of the coil regions in CARPs contained beta and gamma-turn structural motifs. The conformations of the Asp, Glu and Lys hexapeptide or larger CARPs were not defined in the corresponding protein three-dimensional structures analyzed. The longest CARP of known conformation was observed for alanine as a decapeptide in a lysozyme-like protein that corresponds to helix. A feature of CARPs is that a majority are exposed to solvent with accessible surface area greater than 200 ?(2) units in the protein three-dimensional structure.     

Characterization of the active site and thermostability regions of endoxylanase from Thermoanaerobacterium saccharolyticum B6A-RI.

Deletion mutants were constructed from pZEP12, which contained the intact Thermoanaerobacterium saccharolyticum endoxylanase gene (xynA). Deletion of 1.75 kb from the N-terminal end of xynA resulted in a mutant enzyme that retained activity but lost thermostability. Deletion of 1.05 kb from the C terminus did not alter thermostability or activity. The deduced amino acid sequence of T. saccharolyticum B6A-RI endoxylanase XynA was aligned with five other family F beta-glycanases by using the PILEUP program of the Genetics Computer Group package. This multiple alignment of amino acid sequences revealed six highly conserved motifs which included the consensus sequence consisting of a hydrophobic amino acid, Ser or Thr, Glu, a hydrophobic amino acid, Asp, and a hydrophobic amino acid in the catalytic domain. Endoxylanase was inhibited by EDAC [1-(3-dimethylamino propenyl)-3-ethylcarbodiimide hydrochloride], suggesting that Asp and/or Glu was involved in catalysis. Three aspartic acids, two glutamic acids, and one histidine were conserved in all six enzymes aligned. Hydrophobic cluster analysis revealed that two Asp and one Glu occur in the same hydrophobic clusters in T. saccharolyticum B6A-RI endoxylanase and two other enzymes belonging to family F beta-glycanases and suggests their involvement in a catalytic triad. These two Asp and one Glu in XynA from T. saccharolyticum were targeted for analysis by site-specific mutagenesis. Substitution of Asp-537 and Asp-602 by Asn and Glu-600 by Gln completely destroyed endoxylanase activity. These results suggest that these three amino acids form a catalytic triad that functions in a general acid catalysis mechanism.     

Catalytic mechanism of xylose (glucose) isomerase from Clostridium thermosulfurogenes. Characterization of the structural gene and function of active site histidine

The gene coding for thermophilic xylose (glucose) isomerase of Clostridium thermosulfurogenes was isolated and its complete nucleotide sequence was determined. The structural gene (xylA) for xylose isomerase encodes a polypeptide of 439 amino acids with an estimated molecular weight of 50,474. The deduced amino acid sequence of thermophilic C. thermosulfurogenes xylose isomerase displayed higher homology with those of thermolabile xylose isomerases from Bacillus subtilis (70%) and Escherichia coli (50%) than with those of thermostable xylose isomerases from Ampullariella (22%), Arthrobacter (23%), and Streptomyces violaceoniger (24%). Several discrete regions were highly conserved throughout the amino acid sequences of all these enzymes. To identify the histidine residue of the active site and to elucidate its function during enzymatic xylose or glucose isomerization, histidine residues at four different positions in the C. thermosulfurogenes enzyme were individually modified by site-directed mutagenesis. Substitution of His101 by phenylalanine completely abolished enzyme activity whereas substitution of other histidine residues by phenylalanine had no effect on enzyme activity. When His101 was changed to glutamine, glutamic acid, asparagine, or aspartic acid, approximately 10-16% of wild-type enzyme activity was retained by the mutant enzymes. The Gln101 mutant enzyme was resistant to diethylpyrocarbonate inhibition which completely inactivated the wild-type enzyme, indicating that His101 is the only essential histidine residue involved directly in enzyme catalysis. The constant Vmax values of the Gln101, Glu101, Asn101, and Asp101 mutant enzymes over the pH range of 5.0-8.5 indicate that protonation of His101 is responsible for the reduced Vmax values of the wild-type enzyme at pH below 6.5. Deuterium isotope effects by D-[2-2H]glucose on the rate of glucose isomerization indicated that hydrogen transfer and not substrate ring opening is the rate-determining step for both the wild-type and Gln101 mutant enzymes. These results suggest that the enzymatic sugar isomerization does not involve a histidine-catalyzed proton transfer mechanism. Rather, essential histidine functions to stabilize the transition state by hydrogen bonding to the C5 hydroxyl group of the substrate and this enables a metal-catalyzed hydride shift from C2 to C1.     

Intracellular serine protease of Bacillus subtilis: sequence homology with extracellular subtilisins.

Intracellular serine protease was isolated from stationary-grown Bacillus subtilis A-50 cells and purified to homogeneity. The molecular weight of the enzyme is 31,000 +/- 1,000, with an isoelectric point of 4.3. Its amino acid composition is characteristically enriched in glutamic acid content, differing from that of extra-cellular subtilisins. The enzyme is completely inhibited with phenylmethylsulfonyl fluoride and ethylenediaminetetraacetic acid. Intracellular protease possesses negligible activity towards bovine serum albumin and hemoglobin, but has 5- to 20-fold higher specific activity against p-nitroanilides of benzyloxycarbonyl tripeptides than subtilisin BPN'. Esterolytic activity of the enzyme is also higher than that of subtilisin BPN'. The enzyme is sequence homologous with secretory subtilisins throughout 50 determined NH2-terminal residues, indicating the presence of duplicated structural genes for serine proteases in the B. subtilis genome. The occurrence of two homologous genes in the cell might accelerate the evolution of serine protease not only by the loosening of selective constrainst, but also by creation of sequence variants by means of intragenic recombination. Three molecular forms of intracellular protease were found, two of them with NH2-terminal glutamic acid and one minor form, three residues longer, with asparagine as NH2 terminus. These data indicate the possible presence of an enzyme precursor proteolytically modified during cell growth.     

Effect of L-Aspartic Acid and L-Glutamic Acid on Production of L-Proline

To elucidate the effect of aspartic acid on growth of Kurthia catenaforma during the proline fermentation, this organism was compared with other bacteria with respect to the rate of consumption of aspartic acid, and to the activities of enzymes concerned in the metabolism of aspartic acid. Although no marked difference in enzyme activities was observed, the aspartic acid consumption rate of K. catenaforma was markedly higher than that of other organisms. The consumption of glutamic acid by K. catenaforma was not detected at 24 hr of culture. The difference between the consumption of aspartic acid and glutamic acid in this strain might result from a difference in permeability to the amino acids. We considered that L-glutamic acid might substitute for L-aspartic acid if the uptake of glutamic acid could be increased. A number of detergents were screened for their effect on consumption of glutamic acid. Cetyltrimethylammonium bromide, sodium laurylphosphate, and polyoxyethylene sorbitan monolaurate were found to increase the transport rate of glutamic acid, but not of aspartic acid. A method of producing L-proline from glutamic acid was established with the aid of detergents.     

Chemical Modification of Aspartic Acid and Arginine Residues in Horseradish Peroxidase Role of ASP 43 and ARG 38 in the Catalytic Cycle

In an attempt to alter the catalytic properties of horseradish peroxidase (HRP, EC 1.11.1.7), aspartic, glutamic and arginine residues were modified using ethanedithiol and diacetyl. Modification of Asp and Glu led to a marked increase in V_max along with denaturation of the protein. The thiol groups introduced were thought to be responsible, despite being situated on the periphery of the molecule as shown by the modification of the apoenzyme. The role of Arg 38 in the activation of hydrogen peroxide was indicated by the modifications of both enzyme and apoenzyme. An amino acid residue close to Arg 38 was thought to take over its function after blocking the group.     

Purification and characterization of an acidic amino acid specific endopeptidase of Streptomyces griseus obtained from a commercial preparation (Pronase)

A protease was purified 163-fold from Pronase, a commercial product from culture filtrate of Streptomyces griseus, by a series of column chromatographies on CM-Toyopearl (Fractogel), Sephadex G-50, hydroxyapatite, and Z-Gly-D-Phe-AH-Sepharose 4B using Boc-Ala-Ala-Pro-Glu-pNA as a substrate. The final preparation was homogeneous by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and gel isoelectric focusing. Studies on the substrate specificity with peptide p-nitroanilides revealed that this protease preferentially hydrolyzed peptide bonds on the carbonyl-terminal side of either glutamic acid or aspartic acid. It was most active at pH 8.8 for the hydrolysis of Boc-Ala-Ala-Pro-Glu-pNA. The molecular weight of the protease was estimated to be 20,000 by gel filtration on Sepharose 6B using 6 M guanidine hydrochloride as an eluent, and 22,000 by SDS-PAGE in the presence of 2-mercaptoethanol. The isoelectric point of the enzyme was 8.4. The enzyme was inactivated by diisopropyl phosphofluoridate (DFP) but not by p-chloromercuribenzoate (PCMB) or EDTA.     

Site-directed mutagenesis of glutathione S-transferase YaYa. Important roles of tyrosine 9 and aspartic acid 101 in catalysis.

The roles of tyrosine 9 and aspartic acid 101 in the catalytic mechanism of rat glutathione S-transferase YaYa were studied by site-directed mutagenesis. Replacement of tyrosine 9 with phenylalanine (Y9F), threonine (Y9T), histidine (Y9H), or valine (Y9V) resulted in mutant enzymes with less than 5% catalytic activity of the wild type enzymes. Kinetic studies with purified Y9F and Y9T mutants demonstrated poor catalytic efficiencies which were largely due to a drastic decrease in kcat. The estimated pK alpha values of the sulfhydryl group of glutathione bound to Y9F and Y9T mutant enzymes were 8.5 to 8.7, similar to the chemical reaction, in contrast to the estimated pK alpha value of 6.7 to 6.8 for the glutathione enzyme complex of wild type glutathione S-transferase. These results indicate that tyrosine 9 is directly responsible for the lowering of the pKa of the sulfhydryl group of glutathione, presumably due to the stabilization of the thiolate anion through hydrogen bonding with the hydroxyl group of tyrosine. To examine the role of aspartic acid in the binding of glutathione to YaYa, 4 conserved aspartic acid residues at positions 61, 93, 101, and 157 were changed to glutamic acid and asparagine. All mutant enzymes retained either full or partial activity except D157N, which was virtually inactive. Kinetic studies with four mutant enzymes (D93E, D93N, D101E, and D101N) indicate that only D101N exhibited a 5-fold increase in Km toward glutathione. Also, the binding of this mutant to the affinity column was greatly reduced. These results demonstrate that aspartic acid 101 plays an important role in glutathione interaction to YaYa. The role of aspartic acid 157 in catalysis remains to be determined.     

Tb(III) as a fluorescent probe for the structure of bovine serum albumin

Tb(III) was used as a fluorescent probe in the study of the calcium-binding sites on Bovine Serum Albumin (BSA). The fluorescence of Tb(III) is enhanced markedly when bound to BSA and nonradiative energy transfer between two fluorescent tryptophan(Trp) residues and Tb(III) bound to calcium-binding sites on BSA occurred. Experimental results show that the major groups in BSA bound to metal ion are the carboxyl side groups of glutamic acid (Glu) and aspartic acid (Asp). The average distance between the bound Tb(III) and the two tryptophan residues in BSA calculated by a F?ster dipole-dipole nonradiative energy transfer mechanism is 1.48 nm.     

Vitamin K-dependent carboxylation. In vitro modification of synthetic peptides containing the gamma-carboxylation recognition site

Synthetic peptides including the gamma-carboxylation recognition site and acidic amino acids were compared as substrates for vitamin K-dependent gamma-carboxylation by bovine liver carboxylase. The 28-residue proPT28 (proprothrombin -18 to +10) and proFIX28 (pro-Factor IX -18 to +10) were carboxylated with a Km of 3 microM. The Vmax of proPT28 was 2-3 times greater than that of proFIX28. An analog of proFIX28 that contained the prothrombin propeptide had a Vmax 2-3-fold greater than an analog of proPT28 that contained the Factor IX propeptide. proFIX28/RS-1, based upon Factor IX Cambridge, proFIX28/RQ-4, based upon Factor IX Oxford 3, and proFIX28 had equivalent Km and Vmax values. Analogs of proPT28 containing Ala6-Glu7 or Glu6-Ala7 were carboxylated at equivalent rates. A peptide containing Asp6-Asp7 was carboxylated at a rate of about 1% of that of Glu carboxylation. Carboxylation of peptides containing Asp6-Glu7 and Glu6-Asp7 yielded results identical with peptides containing Ala6-Glu7 and Glu6-Ala7. Carboxymethylcysteine was not carboxylated when substituted for Glu6 in a peptide containing Asp7. These results indicate that the prothrombin propeptide is more efficient in the carboxylation process than is the Factor IX propeptide, but that both propeptides direct carboxylation; the gamma-carboxylation recognition site does not include residues -4 and -1; aspartic acid and carboxymethylcysteine are poor substrates for the carboxylase, but aspartic acid does not inhibit the carboxylation of adjacent glutamic acids.     

Effect of propeptide mutations on post-translational processing of factor IX. Evidence that beta-hydroxylation and gamma-carboxylation are independent events

Post-translational processing of Factor IX includes glycosylation, cleavage of the signal peptide and propeptide, vitamin K-dependent carboxylation of specific glutamic acid residues to form gamma-carboxyglutamic acid, and beta-hydroxylation of aspartic acid at residue 64 to form beta-hydroxyaspartic acid. The human Factor IX cDNA coding sequence was modified in the propeptide region (residue -18 to -1) using oligonucleotide-directed site-specific mutagenesis, and the altered Factor IX cDNA was expressed in Chinese hamster ovary cells. The effects of the mutations on proteolytic processing, gamma-carboxylation, and beta-hydroxylation were assessed by direct structural analysis. After purification, the molecular weight of each of the recombinant Factor IX species and its NH2-terminal amino acid sequence were shown to be identical to those of plasma Factor IX. gamma-Carboxyglutamic acid and beta-hydroxyaspartic acid analyses revealed that recombinant wild-type Factor IX contained 9.2 gamma-carboxyglutamic acid and 0.3 beta-hydroxyaspartic acid residues/molecule compared with 11.4 gamma-carboxyglutamic acid and 0.39 beta-hydroxyaspartic acid residues in plasma Factor IX. When the 18-residue propeptide was deleted or when the cells were grown in the presence of sodium warfarin, secreted Factor IX contained no detectable gamma-carboxyglutamic acid but 0.36 and 0.40 residues of beta-hydroxyaspartic acid, respectively. Point mutations leading to substitution of alanine for phenylalanine at residue -16 or glutamic acid for alanine at residue -10 contained 0.2 and 1.7 gamma-carboxyglutamic acid residues, respectively, and 0.2 residues of beta-hydroxyaspartic acid. These data confirm that the propeptide mutations made do not interfere with proteolytic processing and that the Factor IX propeptide contains a recognition site that designates the adjacent glutamic acid-rich domain for gamma-carboxylation. In contrast, beta-hydroxylation of aspartic acid 64 is an independent process which does not require vitamin K and is mediated through a hydroxylation recognition site in the mature Factor IX, not in the propeptide.