OmpT: molecular dynamics simulations of an outer membrane enzyme

Five molecular dynamics simulations (total duration >25 ns) have been performed on the Escherichia coli outer membrane protease OmpT embedded in a dimyristoylphosphatidylcholine lipid bilayer. Globally the protein is conformationally stable. Some degree of tilt of the beta-barrel is observed relative to the bilayer plane. The greatest degree of conformational flexibility is seen in the extracellular loops. A complex network of fluctuating H-bonds is formed between the active site residues, such that the Asp210-His212 interaction is maintained throughout, whereas His212 and Asp83 are often bridged by a water molecule. This supports a catalytic mechanism whereby Asp83 and His212 bind a water molecule that attacks the peptide carbonyl. A configuration yielded by docking calculations of OmpT simulation snapshots and a model substrate peptide Ala-Arg-Arg-Ala was used as the starting point for an extended Huckel calculation on the docked peptide. These placed the lowest unoccupied molecular orbital mainly on the carbon atom of the central C=O in the scissile peptide bond, thus favoring attack on the central peptide by the water held by residues Asp83 and His212. The trajectories of water molecules reveal exchange of waters between the intracellular face of the membrane and the interior of the barrel but no exchange at the extracellular mouth. This suggests that the pore-like region in the center of OmpT may enable access of water to the active site from below. The simulations appear to reveal the presence of specific lipid interaction sites on the surface of the OmpT barrel. This reveals the ability of extended MD simulations to provide meaningful information on protein-lipid interactions.     

Contribution of active site residues to substrate hydrolysis by USP2: insights into catalysis by ubiquitin specific proteases

The ubiquitin-specific protease (USP) structural class represents the largest and most diverse family of deubiquitinating enzymes (DUBs). Many USPs assume important biological roles and emerge as potential targets for therapeutic intervention. A clear understanding of USP catalytic mechanism requires a functional evaluation of the proposed key active site residues. Crystallographic data of ubiquitin aldehyde adducts of USP catalytic cores provided structural details on the catalytic triad residues, namely the conserved Cys and His, and a variable putative third residue, and inferred indirect structural roles for two other conserved residues (Asn and Asp), in stabilizing via a bridging water molecule the oxyanion of the tetrahedral intermediate (TI). We have expressed the catalytic domain of USP2 and probed by site-directed mutagenesis the role of these active site residues in the hydrolysis of peptide and isopeptide substrates, including a synthetic K48-linked diubiquitin substrate for which a label-free, mass spectrometry based assay has been developed to monitor cleavage. Hydrolysis of ubiquitin-AMC, a model substrate, was not affected by the mutations. Molecular dynamics simulations of USP2, free and complexed with the TI of a bona fide isopeptide substrate, were carried out. We found that Asn271 is structurally poised to directly stabilize the oxyanion developed in the acylation step, while being structurally supported by the adjacent absolutely conserved Asp575. Mutagenesis data functionally confirmed this structural role independent of the nature (isopeptide vs peptide) of the bond being cleaved. We also found that Asn574, structurally located as the third member of the catalytic triad, does not fulfill this role functionally. A dual supporting role is inferred from double-point mutation and structural data for the absolutely conserved residue Asp575, in oxyanion hole formation, and in maintaining the correct alignment and protonation of His557 for catalytic competency.     

Identification of essential catalytic residues of the cyclase NisC involved in the biosynthesis of nisin

Nisin is a post-translationally modified antimicrobial peptide that has been widely used in the food industry for several decades. It contains five cyclic thioether cross-links of varying sizes that are installed by a single enzyme, NisC, that catalyzes the addition of cysteines to dehydroamino acids. The recent x-ray crystal structure of NisC has provided the first insights into the catalytic residues responsible for the cyclization step during nisin biosynthesis. In this study, the conserved residues His(212), Arg(280), Asp(141), and Tyr(285) as well as the ligands to the zinc in the active site (Cys(284), Cys(330), and His(331)) were substituted by site-directed mutagenesis. Binding studies showed that all mutants had similar affinities for NisA. Activity assays showed that whereas His(212) and Asp(141) were essential for correct cyclization as judged by the antimicrobial activity of the final product, Arg(280) and Tyr(285) were not. Mutation of zinc ligands to alanine also abolished the enzymatic activity, and these mutant proteins were shown to contain decreased levels of zinc. These results show that the zinc is essential for activity and support a model in which the zinc is used to activate the cysteines in the substrate for nucleophilic attack. These findings also argue against an essential role of Arg(280) and Tyr(285) as an active site general acid/base in the mechanism of cyclization.     

1.68-A crystal structure of endopolygalacturonase II from Aspergillus niger and identification of active site residues by site-directed mutagenesis.

Polygalacturonases specifically hydrolyze polygalacturonate, a major constituent of plant cell wall pectin. To understand the catalytic mechanism and substrate and product specificity of these enzymes, we have solved the x-ray structure of endopolygalacturonase II of Aspergillus niger and we have carried out site-directed mutagenesis studies. The enzyme folds into a right-handed parallel beta-helix with 10 complete turns. The beta-helix is composed of four parallel beta-sheets, and has one very small alpha-helix near the N terminus, which shields the enzyme's hydrophobic core. Loop regions form a cleft on the exterior of the beta-helix. Site-directed mutagenesis of Asp(180), Asp(201), Asp(202), His(223), Arg(256), and Lys(258), which are located in this cleft, results in a severe reduction of activity, demonstrating that these residues are important for substrate binding and/or catalysis. The juxtaposition of the catalytic residues differs from that normally encountered in inverting glycosyl hydrolases. A comparison of the endopolygalacturonase II active site with that of the P22 tailspike rhamnosidase suggests that Asp(180) and Asp(202) activate the attacking nucleophilic water molecule, while Asp(201) protonates the glycosidic oxygen of the scissile bond.     

Involvement of the Arg-Asp-His catalytic triad in enzymatic cleavage of the phosphodiester bond

Phosphatidylinositol-specific phospholipase C (PI-PLC) catalyzes the cleavage of the P-O bond in phosphatidylinositol via intramolecular nucleophilic attack of the 2-hydroxyl group of inositol on the phosphorus atom. Our earlier stereochemical and site-directed mutagenesis studies indicated that this reaction proceeds by a mechanism similar to that of RNase A, and that the catalytic site of PI-PLC consists of three major components analogous to those observed in RNase A, the His32 general base, the His82 general acid, and Arg69 acting as a phosphate-activating residue. In addition, His32 is associated with Asp274 in forming a catalytic triad with inositol 2-hydroxyl, and His82 is associated with Asp33 in forming a catalytic diad. The focus of this work is to provide a global view of the mechanism, assess cooperation between various catalytic residues, and determine the origin of enzyme activation by the hydrophobic leaving group. To this end, we have investigated kinetic properties of Arg69, Asp33, and His82 mutants with phosphorothioate substrate analogues which feature leaving groups of varying hydrophobicity and pK(a). Our results indicate that interaction of the nonbridging pro-S oxygen atom of the phosphate group with Arg69 is strongly affected by Asp33, and to a smaller extent by His82. This result in conjunction with those obtained earlier can be rationalized in terms of a novel, dual-function triad comprised of Arg69, Asp33, and His82 residues. The function of this triad is to both activate the phosphate group toward the nucleophilic attack and to protonate the leaving group. In addition, Asp33 and His82 mutants displayed much smaller degrees of activation by the fatty acid-containing leaving group as compared to the wild-type (WT) enzyme, and the level of activation was significantly reduced for substrates featuring the leaving group with low pK(a) values. These results strongly suggest that the assembly of the above three residues into the fully catalytically competent triad is controlled by the hydrophobic interactions of the enzyme with the substrate leaving group.     

Study on the hydrolysis mechanism of phosphodiesterase 4 using molecular dynamics simulations

We carried out NPT molecular dynamics simulations in an explicit solvent to better understand the mechanism of cyclic adenosine monophosphates (cAMP) hydrolysis by phosphodiesterase 4 (PDE4) enzyme on atomic details and to obtain information on the dynamics characteristic of the catalytic domains of PDE4. In analyzing the water hydrogen-bond network around the active site, we also showed the importance of water in drug–protein interactions. In addition, we reported the characteristics of the hydration pattern and the dynamic distance distribution around the interesting residues. The results indicated that Asp318 plays the role of a general base that can activate water molecule for nucleophilic attack on cAMP. As expected, His160 plays the role of a proton donor for cAMP.     

Functional analysis of conserved aspartate and histidine residues located around the type 2 copper site of copper-containing nitrite reductase

A heterologous expression system of the blue copper-containing nitrite reductase from Alcaligenes xylosoxidans GIFU1051 (AxgNIR) was constructed, and the purified recombinant enzyme was characterized. All the characteristic spectroscopic properties and enzyme activity of native AxgNIR were retained in the copper-reconstituted recombinant protein expressed in Escherichia coli, indicating the correct coordination of two types of Cu (type 1 and 2) in the recombinant enzyme. Moreover, two conserved noncoordinate residues, Asp98 and His255, located near the type 2 Cu site were replaced to elucidate the catalytic residue(s) of NIR. The Asp98 residue hydrogen-bonded to the water molecule ligating the type 2 Cu was changed to Ala, Asn, or Glu, and the His255 residue hydrogen-bonded to Asp98 through the water molecule was replaced with Ala, Lys, or Arg. The catalytic rate constants of all mutants were decreased to 0.4-2% of those of the recombinant enzyme, and the apparent K(m) values for nitrite were greatly increased in the Asp98 mutants. All the steady-state kinetic data of the mutants clearly demonstrate that both Asp98 and His255 are involved not only in the catalytic reaction but also in the substrate anchoring.     

Sequence of the subtilisin-encoding gene from an antarctic psychrotroph Bacillus TA41.

The nucleotide sequence of the subtilisin-encoding gene from the antarctic psychrotroph, Bacillus TA41, was determined. The primary structure of the subtilisin precursor corresponds to a preproenzyme of 419 amino acids. Asp144, His181 and Ser359 are the proposed catalytic residues of the protease active site.     

Mutation of a unique aspartate residue abolishes the catalytic activity but not substrate binding of the mouse N-methylpurine-DNA glycosylase (MPG)

N-Methylpurine-DNA glycosylase (MPG) initiates base excision repair in DNA by removing a variety of alkylated purine adducts. Although Asp was identified as the active site residue in various DNA glycosylases based on the crystal structure, Glu-125 in human MPG (Glu-145 in mouse MPG) was recently proposed to be the catalytic residue. Mutational analysis for all Asp residues in a truncated, fully active MPG protein showed that only Asp-152 (Asp-132 in the human protein), which is located near the active site, is essential for catalytic activity. However, the substrate binding was not affected in the inactive Glu-152, Asn-152, and Ala-152 mutants. Furthermore, mutation of Asp-152 did not significantly affect the intrinsic tryptophan fluorescence of the enzyme and the far UV CD spectra, although a small change in the near UV CD spectra of the mutants suggests localized conformational change in the aromatic residues. We propose that in addition to Glu-145 in mouse MPG, which functions as the activator of a water molecule for nucleophilic attack, Asp-152 plays an essential role either by donating a proton to the substrate base and, thus, facilitating its release or by stabilizing the steric configuration of the active site pocket.     

Catalysis-linked inactivation of fluoroacetate dehalogenase by ammonia: a novel approach to probe the active-site environment

Fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX) catalyzes the hydrolytic dehalogenation of fluoroacetate and other haloacetates. Asp(105) of the enzyme acts as a nucleophile to attack the alpha-carbon of haloacetate to form an ester intermediate, which is subsequently hydrolyzed by a water molecule activated by His(272) [Liu, J.Q., Kurihara, T., Ichiyama, S., Miyagi, M., Tsunasawa, S., Kawasaki, H., Soda, K., and Esaki, N. (1998) J. Biol. Chem. 273, 30897-30902]. In this study, we found that FAc-DEX is inactivated concomitantly with defluorination of fluoroacetate by incubation with ammonia. Mass spectrometric analyses revealed that the inactivation of FAc-DEX is caused by nucleophilic attack of ammonia on the ester intermediate to convert the catalytic residue, Asp(105), into an asparagine residue. The results indicate that ammonia reaches the active site of FAc-DEX without losing its nucleophilicity. Analysis of the three-dimensional structure of the enzyme by homology modeling showed that the active site of the enzyme is mainly composed of hydrophobic and basic residues, which are considered to be essential for an ammonia molecule to retain its nucleophilicity. In a normal enzyme reaction, the hydrophobic environment is supposed to prevent hydration of the highly electronegative fluorine atom of the substrate and contribute to fluorine recognition by the enzyme. Basic residues probably play a role in counterbalancing the electronegativity of the substrate. These results demonstrate that catalysis-linked inactivation is useful for characterizing the active-site environment as well as for identifying the catalytic residue.     

A molecular dynamics study of the structural stability of HIV-1 protease under physiological conditions: the role of Na+ ions in stabilizing the active site

HIV-1 protease is most active under weakly acidic conditions (pH 3.5-6.5), when the catalytic Asp25 and Asp25' residues share 1 proton. At neutral pH, this proton is lost and the stability of the structure is reduced. Here we present an investigation of the effect of pH on the dynamics of HIV-1 protease using MD simulation techniques. MD simulations of the solvated HIV-1 protease with the Asp25/25' residues monoprotonated and deprotonated have been performed. In addition we investigated the effect of the inclusion of Na(+) and Cl(-) ions to mimic physiological salt conditions. The simulations of the monoprotonated form and deprotonated form including Na(+) show very similar behavior. In both cases the protein remained stable in the compact, "self-blocked" conformation in which the active site is blocked by the tips of the flaps. In the deprotonated system a Na(+) ion binds tightly to the catalytic dyad shielding the repulsion between the COO(-) groups. Ab initio calculations also suggest the geometry of the active site with the Na(+) bound closely resembles that of the monoprotonated case. In the simulations of the deprotonated form (without Na(+) ions), a water molecule bound between the Asp25 Asp25' side-chains. This disrupted the dimerization interface and eventually led to a fully open conformation.     

The complete amino acid sequence of rat submaxillary gland tonin does contain the aspartic acid at the active site: confirmation by protein sequence analysis

The revised amino acid sequence of rat submaxillary gland tonin, a serine protease, does contain the active site Asp residue. The active site of this kallikrein-related enzyme is thus made up of the same catalytic triad (Asp, Ser, and His) found in all known serine proteases. The important Asp residue has now been localized in a 16 amino acid peptide previously reported as missing in the tonin sequence. The complete amino acid sequence thus contains 235 residues corresponding to a molecular weight of 25,658, more in agreement with previously reported molecular weights. Moreover, the revised structure led (a) to the assignment of Arg, Asn, and Val residues instead of His, Asp, and Gly at positions 63, 165, and 169, respectively; (b) to the assignment of residues occupying an overlapping sequence at positions 165-171, and finally (c) to the localization of two N-glycosylation sites at positions 82 and 165. These results further document the close relationship of tonin to the ever expanding kallikrein family.     

A QM/QM investigation of the hUNG2 reaction surface: the untold tale of a catalytic residue

Human uracil-DNA glycosylase (hUNG2) is a base excision repair enzyme that removes the damaged base uracil from DNA through hydrolytic deglycosylation of the nucleotide. In the present study, the mechanism of hUNG2 is thoroughly investigated using ONIOM(MPWB1K/6-31G(d):PM3) active-site models to generate reaction potential energy surfaces. Active-site models that differ in the hydrogen-bonding arrangement of the nucleophilic water molecule and/or protonation state of His148 are considered. The large barrier calculated using the model with a cationic His148 verifies that this residue is neutral in the early stages of the reaction. The reaction pathways predicted by two models with a neutral His148 are consistent with a wealth of experimental data on the enzyme, including mutational studies, which supports our approach. On the basis of our calculations, we propose a complete mechanism for the chemical step of hUNG2. In the first part of the reaction, His268, Asn204, and a water molecule work together to stabilize the negative charge forming on the uracil moiety. Subsequently, either Asp145 or His148 can act as the general base that activates the water nucleophile depending on the binding orientation of the water molecule in the active site. However, we propose that His148 preferentially acts as the general base. Therefore, in agreement with previous proposals, we assign the primary function of Asp145 to electrostatic stabilization of the positive charge developing on the sugar moiety during the reaction, which is also consistent with a growing theory that the primary function of active-site carboxylate groups present in many glycosylases is transition state stabilization. Most importantly, our work explains, for the first time, the role of His148 in the chemical step and provides additional support for the inclusion of this amino acid in the list of residues (Asp145 and His268) essential to the chemical step of the hUNG2 mechanism.     

Putative reaction mechanism of heterologously expressed octopine dehydrogenase from the great scallop, Pecten maximus (L)

cDNA for octopine dehydrogenase (ODH) from the adductor muscle of the great scallop, Pecten maximus, was cloned using 5'- and 3'-RACE. The cDNA comprises an ORF of 1197 nucleotides and the deduced amino acid sequence encodes a protein of 399 amino acids. ODH was heterologously expressed in Escherichia coli with a C-terminal penta His-tag. ODH-5His was purified to homogeneity using metal-chelate affinity chromatography and Sephadex G-100 gel filtration. Recombinant ODH had kinetic properties similar to those of wild-type ODH isolated from the scallop's adductor muscle. Site-directed mutagenesis was used to elucidate the involvement of several amino acid residues for the reaction catalyzed by ODH. Cys148, which is conserved in all opine dehydrogenases known to date, was converted to serine or alanine, showing that this residue is not intrinsically important for catalysis. His212, Arg324 and Asp329, which are also conserved in all known opine dehydrogenase sequences, were subjected to site-directed mutagenesis. Modification of these residues revealed their importance for the catalytic activity of the enzyme. Conversion of each of these residues to alanine resulted in strong increases in K(m) and decreases in k(cat) values for pyruvate and L-arginine, but had little effect on the K(m) and k(cat) values for NADH. Assuming a similar structure for ODH compared with the only available structure of a bacterial opine dehydrogenase, these three amino acids may function as a catalytic triad in ODH similar to that found in lactate dehydrogenase or malate dehydrogenase. The carboxyl group of pyruvate is then stabilized by Arg324. In addition to orienting the substrate, His212 will act as an acid-base catalyst by donating a proton to the carbonyl group of pyruvate. The acidity of this histidine is further increased by the proximity of Asp329.     

Structural insights into the activation and inhibition of histo-aspartic protease from Plasmodium falciparum

Histo-aspartic protease (HAP) from Plasmodium falciparum is a promising target for the development of novel antimalarial drugs. The sequence of HAP is highly similar to those of pepsin-like aspartic proteases, but one of the two catalytic aspartates, Asp32, is replaced with histidine. Crystal structures of the truncated zymogen of HAP and of the complex of the mature enzyme with inhibitor KNI-10395 have been determined at 2.1 and 2.5 ? resolution, respectively. As in other proplasmepsins, the propeptide of the zymogen interacts with the C-terminal domain of the enzyme, forcing the N- and C-terminal domains apart, thereby separating His32 and Asp215 and preventing formation of the mature active site. In the inhibitor complex, the enzyme forms a tight domain-swapped dimer, not previously seen in any aspartic proteases. The inhibitor is found in an unprecedented conformation resembling the letter U, stabilized by two intramolecular hydrogen bonds. Surprisingly, the location and conformation of the inhibitor are similar to those of the fragment of helix 2 comprising residues 34p-38p in the prosegments of the zymogens of gastric aspartic proteases; a corresponding helix assumes a vastly different orientation in proplasmepsins. Each inhibitor molecule is in contact with two molecules of HAP, interacting with the carboxylate group of the catalytic Asp215 of one HAP protomer through a water molecule, while also making a direct hydrogen bond to Glu278A' of the other protomer. A comparison of the shifts in the positions of the catalytic residues in the inhibitor complex presented here with those published previously gives further hints regarding the enzymatic mechanism of HAP.     

His ... Asp catalytic dyad of ribonuclease A: histidine pKa values in the wild-type, D121N, and D121A enzymes

Bovine pancreatic ribonuclease A (RNase A) has a conserved His ... Asp catalytic dyad in its active site. Structural analyses had indicated that Asp121 forms a hydrogen bond with His119, which serves as an acid during catalysis of RNA cleavage. The enzyme contains three other histidine residues including His12, which is also in the active site. Here, 1H-NMR spectra of wild-type RNase A and the D121N and D121A variants were analyzed thoroughly as a function of pH. The effect of replacing Asp121 on the microscopic pKa values of the histidine residues is modest: none change by more than 0.2 units. There is no evidence for the formation of a low-barrier hydrogen bond between His119 and either an aspartate or an asparagine residue at position 121. In the presence of the reaction product, uridine 3'-phosphate (3'-UMP), protonation of one active-site histidine residue favors protonation of the other. This finding is consistent with the phosphoryl group of 3'-UMP interacting more strongly with the two active-site histidine residues when both are protonated. Comparison of the titration curves of the unliganded enzyme with that obtained in the presence of different concentrations of 3'-UMP shows that a second molecule of 3'-UMP can bind to the enzyme. Together, the data indicate that the aspartate residue in the His ... Asp catalytic dyad of RNase A has a measurable but modest effect on the ionization of the adjacent histidine residue.     

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.     

Active site binding modes of curcumin in HIV-1 protease and integrase

Structure models for the interaction of curcumin with HIV-1 integrase (IN) and protease (PR) were investigated using computational docking. Curcumin was found to bind preferentially in similar ways to the active sites of both IN and PR. For IN, the binding site is formed by residues Asp64, His67, Thr66, Glu92, Thr93, Asp116, Ser119, Asn120, and Lys159. Docked curcumin contacts the catalytic residues adjacent to Asp116 and Asp64, and near the divalent metal (Mg2+). In the PR docking, the curcumin structure fitted well to the active site, interacting with residues Asp25, Asp29, Asp30, Gly27', Asp29', and Asp30'. The results suggest that o-hydroxyl and/or keto-enol structures are important for both IN and PR inhibitory actions. The symmetrical structure of curcumin seems to play an important role for binding to the PR protein, whereas the keto-enol and only one side of the terminal o-hydroxyl showed tight binding to the IN active site.     

Identification of active site serine and histidine residues in Escherichia coli outer membrane protease OmpT

Escherichia coli outer membrane protease OmpT has been characterised as a serine protease based on its inhibitor profile, but serine protease consensus sequences are absent. By site-directed mutagenesis we substituted all conserved serines and histidines. Substitution of His(101) and His(212) by Ala, Asn or Gln resulted in variant enzymes with 0.01 and 9-20% residual enzymatic activity towards a fluorogenic pentapeptide substrate, respectively. The mutations S140A and S201A did not decrease activity, while variants S40A and S99A yielded 0.5 and 0.2% residual activities, respectively. When measured with a dipeptide substrate the variant S40A demonstrated full activity, whereas variant S99A displayed at least 500-fold reduced activity. We conclude that Ser(99) and His(212) are essential active site residues. We propose that OmpT is a novel serine protease with Ser(99) as the active site nucleophile and His(212) as general base.     

Rat liver 10-formyltetrahydrofolate dehydrogenase, carbamoyl phosphate synthetase 1 and betaine homocysteine S-methytransferase were co-purified on Kunitz-type soybean trypsin inhibitor-coupled sepharose CL-4B

An Asp/His catalytic site of 10-formyltetrahydrofolate dehydrogenase (FDH) was suggested to have a similar catalytic topology with the Asp/His catalytic site of serine proteases. Many studies supported the hypothesis that serine protease inhibitors can bind and modulate the activity of serine proteases by binding to the catalytic site of serine proteases. To explore the possibility that soybean trypsin inhibitor (SBTI) can recognize catalytic sites of FDH and can make a stable complex, we carried out an SBTI-affinity column by using rat liver homogenate. Surprisingly, the Rat FDH molecule with two typical liver proteins, carbamoyl-phosphate synthetase 1 (CPS1) and betaine homocysteine S-methyltransferase (BHMT) were co-purified to homogeneity on SBTI-coupled Sepharose and Sephacryl S-200 followed by Superdex 200 FPLC columns. These three liver-specific proteins make a protein complex with 300 kDa molecular mass on the gel-filtration column chromatography in vitro. Immuno-precipitation experiments by using anti-FDH and anti-SBTI antibodies also supported the fact that FDH binds to SBTI in vitro and in vivo. These results demonstrate that the catalytic site of rat FDH has a similar structure with those of serine proteases. Also, the SBTI-affinity column will be useful for the purification of rat liver proteins such as FDH, CPS1 and BHMT.