The HELLGH motif of rat liver dipeptidyl peptidase III is involved in zinc coordination and the catalytic activity of the enzyme.

The role of the HELLGH (residues 450-455) motif in the sequence of rat dipeptidyl peptidase III (EC 3.4.14.4) was investigated by replacing Glu451 with an alanine or an aspartic acid residue and by replacing His450 and His455 with a tyrosine residue by site-directed mutagenesis. Mutated cDNAs were expressed three or four times in Escherichia coli, and the resulting proteins were purified to apparent homogeneity. None of the expressed mutated proteins exhibited DPP III activity. The mutants of Glu451 contained 1 mol of zinc per mole of protein, but mutants His450 and His455 did not contain significant amounts of zinc as determined by atomic absorption spectrometry. The Leu453-deleted enzyme (having the zinc aminopeptidase motif HExxH-18-E) had almost the same order of binding affinity (for Arg-Arg-2-naphthylamide) as the wild-type enzyme, but the specificity constant was about 10%. These results provide evidence that the suitable number of amino acids included between Glu451 and His455 is three residues for the enzyme activity and confirm that residues His450, His455, and Glu451 are involved in zinc coordination and catalytic activity.     

Identification of the reactive cysteine residues in yeast dipeptidyl peptidase III

Dipeptidyl peptidases III (DPPs III) form a distinct metallopeptidase family characterized by the unique HEXXGH motif. High susceptibility to inactivation by organomercurials suggests the presence of a reactive cysteine residue(s) in, or close to, their active site. Yeast DPP III contains five Cys, none of which is absolutely conserved within the family. In order to identify reactive residue(s), site-directed mutagenesis on yeast His₆-tagged DPP III was employed to substitute specifically all five cysteine residues to serine. The variant enzymes thus obtained were enzymatically active and showed an overall structure not greatly affected by the mutations as judged by circular dichroism. Analysis by native and SDS-PAGE under non-reducing conditions revealed the existence of a monomeric and dimeric form in all DPP III proteins except in the C130S, implying that dimerization of yeast DPP III is mediated by the surface-exposed cysteine 130.     

Purification and characterization of human uroporphyrinogen III synthase expressed in Escherichia coli

The side-chain asymmetry of physiological porphyrins is produced by the cooperative action of hydroxymethylbilane synthase and uroporphyrinogen (uro'gen) III synthase. Although the role of uro'gen III synthase is essential for the chemistry of porphyrin biosynthesis, many aspects, structural as well as mechanical, of uro'gen III synthase have yet to be studied. We report here an expression system in Escherichia coli and a purification procedure for human uro'gen III synthase. The enzyme in the lysate was unstable, but we found that glycerol prevents the activity loss in the lysate. The purified enzyme showed remarkable thermostability, particularly when kept in phosphate buffer containing DTT or EDTA, indicating that the enzyme activity may depend on its oxidation state. Examination of the relationship between the number of Cys residues that are accessible to 5,5'-dithiobis(2-nitrobenzoic acid) and the remaining activity during heat inactivation showed that a particular Cys residue is involved in activity loss. From the crystal structure of human uro'gen III synthase [Mathews et al. (2001) EMBO J. 20, 5832-5839], this Cys residue was considered to be Cys73, which is buried deep inside the enzyme, suggesting that Cys73 of human uro'gen III synthase plays an important role in enzyme activity.     

The crystal structure of a sulfurtransferase from Azotobacter vinelandii highlights the evolutionary relationship between the rhodanese and phosphatase enzyme families

Rhodanese is an ubiquitous enzyme that in vitro catalyses the transfer of a sulfur atom from suitable donors to nucleophilic acceptors by way of a double displacement mechanism. During the catalytic process the enzyme cycles between a sulfur-free and a persulfide-containing form, via formation of a persulfide linkage to a catalytic Cys residue. In the nitrogen-fixing bacteria Azotobacter vinelandii the rhdA gene has been identified and the encoded protein functionally characterized as a rhodanese. The crystal structure of the A. vinelandii rhodanese has been determined and refined at 1.8 A resolution in the sulfur-free and persulfide-containing forms. Conservation of the overall three-dimensional fold of bovine rhodanese is observed, with substantial modifications of the protein structure in the proximity of the catalytic residue Cys230. Remarkably, the native enzyme is found as the Cys230-persulfide form; in the sulfur-free state the catalytic Cys residue adopts two alternate conformations, reflected by perturbation of the neighboring active-site residues, which is associated with a partly reversible loss of thiosulfate:cyanide sulfurtransferase activity. The catalytic mechanism of A. vinelandii rhodanese relies primarily on the main-chain conformation of the 230 to 235 active-site loop and on a surrounding strong positive electrostatic field. Substrate recognition is based on residues which are entirely different in the prokaryotic and eukaryotic enzymes. The active-site loop of A. vinelandii rhodanese displays striking structural similarity to the active-site loop of the similarly folded catalytic domain of dual specific phosphatase Cdc25, suggesting a common evolutionary origin of the two enzyme families.     

Human and rat dipeptidyl peptidase III: biochemical and mass spectrometric arguments for similarities and differences

Dipeptidyl peptidase (DPP III) was purified from rat and human erythrocytes using an identical procedure. Electrophoretic analyses revealed the same molecular size and pI for both enzymes. The molecular mass of the human enzyme, measured by matrix-assisted laser desorption/ionization MS, was 82500+/-60 Da. Its tryptic peptide mass profile was determined using the same technique, and the amino acid sequence of two internal peptides was obtained by tandem MS and Edman degradation. A search of databases revealed a high similarity between the human erythrocyte and rat liver DPP III: 21 matches out of 34 detected peptides were found, covering 40% of the total sequence. Matched peptides included the peptide harboring the characteristic HELLGH sequence motif, and a stretch of 19 identical amino acids, containing Glu, a putative ligand of active site zinc. Both enzymes preferred Arg-Arg-2-naphthylamide, and were activated by micromolar Co2+, differing in their pH optima and kcat/Km. Zn2+ ions, sulfhydryl reagents, and aminopeptidase inhibitors, especially probestin, inhibited the rat DPP III more potently. The two enzymes showed the highest affinity for angiotensin III (Ki < 1 microM) and a preference for ahydrophobic residue at the P1' site. However, significant differences in the binding constants for several peptides indicated non-identity in the active site topography of human and rat erythrocyte DPP III.     

Absolutely conserved tryptophan in M49 family of peptidases contributes to catalysis and binding of competitive inhibitors

The role of the unique fully conserved tryptophan in metallopeptidase family M49 (dipeptidyl peptidase III family) was investigated by site-directed mutagenesis on human dipeptidyl peptidase III (DPP III) where Trp300 was subjected to two substitutions (W300F and W300L). The mutant enzymes showed thermal stability equal to the wild-type DPP III. Conservative substitution of the Trp300 with phenylalanine decreased enzyme activity 2-4 fold, but did not significantly change the K_m values for two dipeptidyl 2-naphthylamide substrates. However, the K_m for the W300L mutant was elevated 5-fold and the k_cat value was reduced 16-fold with Arg-Arg-2-naphthylamide. Both substitutions had a negative effect on the binding of two competitive inhibitors designed to interact with S1 and S2 subsites.These results indicate the importance of the aromatic nature of W300 in DPP III ligand binding and catalysis, and contribution of this residue in maintaining the functional integrity of this enzyme’s S2 subsite.     

Flexibility of the coordination geometry around the cupric ions in Cu(II)-rat dipeptidyl peptidase III is important for the expression of enzyme activity

Dipeptidyl peptidase III (DPP III), the zinc peptidase, has a unique helix portion in the metal-binding motif (HELLGH). The enzyme activity of the cupric derivative of rat DPP III (Cu(II)-rat DPP III) for Lys-Ala-β-NA is about 30% of that of the wild-type enzyme. On the other hand, the enzyme activity of Cu(II)-rat del-DPP III, in which Leu453 is deleted from the metal-binding motif, possesses only 1-2% of the enzyme activity of rat del-DPP III. The EPR spectra of Cu(II)-rat DPP III in the presence of various concentrations of the substrate, Lys-Ala-β-NA, changed dramatically, showing formation of the enzyme-metal-substrate complex. The EPR spectra of Cu(II)-rat del-DPP III did not change in the presence of excess Lys-Ala-β-NA. The deletion of Leu453 from the HELLGH motif of rat DPP III leads to a complete loss of flexibility in the ligand geometry around the cupric ions. Under the formation of the enzyme-metal-substrate complex, Glu451 of Cu(II)-rat DPP III is sufficiently able to approach the water molecule via a very different orientation from that of the resting state; however, Glu451 of Cu(II)-rat del-DPP III is not able to access the water molecule.     

Highly reactive cysteine residues are part of the substrate binding site of mammalian dipeptidyl peptidases III

Dipeptidyl peptidase III (DPP III) is a cytosolic zinc-exopeptidase involved in the intracellular protein catabolism of eukaryotes. Although inhibition by thiol reagents is a general feature of DPP III originating from various species, the function of activity important sulfhydryl groups is still inadequately understood. The present study of the reactivity of these groups was undertaken in order to clarify their biological significance.The inactivation kinetics of human and rat DPP III by sulfhydryl reagent p-hydroxy-mercuribenzoate (pHMB) was monitored by determination of the enzyme's residual activity with fluorimetric detection.Inactivation of this human enzyme exhibited pseudo-first-order kinetics, suggesting that all reactive SH-groups have equivalent reactivity, and the second-order rate constant was calculated to be 3523+/-567M(-1)min(-1). Rat DPP III was hyperreactive to pHMB and showed biphasic kinetics indicating two classes of reactive SH-groups. The second-order rate constants of 3540M(-1)s(-1) for slower reacting sulfhydryl, and 21,855M(-1)s(-1) for faster reacting sulfhydryl were obtained from slopes of linear plots of pseudo-first-order constants versus reagent concentration. Peptide substrates protected both mammalian DPPs III from inactivation by pHMB. Physiological concentrations of biological thiols and H(2)O(2) inactivated the rat DPP III. Human enzyme was resistant to H(2)O(2) attack and less affected by reduced glutathione (GSH) than the rat homologue. A significantly lower DPP III level, determined by activity measurement and Western blotting, was found in the cytosols of highly oxygenated rat tissues.These results provide kinetic evidence that cysteine residues are involved in substrate binding of mammalian DPPs III.     

Functional tyrosine residue in the active center of human dipeptidyl peptidase III

Abstract Human dipeptidyl peptidase III (DPP III) is a member of the metallopeptidase family M49 with an implied role in the pain-modulatory system and endogenous defense against oxidative stress. Here, we report the heterologous expression of human DPP III and the site-directed mutagenesis results which demonstrate a functional role for Tyr318 at the active site of this enzyme. The substitution of Tyr318 to Phe decreased kcat by two orders of magnitude without altering the binding affinity of substrate, or of a competitive hydroxamate inhibitor designed to interact with S1 and S2 subsites. The results indicate that the conserved tyrosine could be involved in transition state stabilization during the catalytic action of M49 peptidases.     

Characterization of the metal-substituted dipeptidyl peptidase III (rat liver)

Dipeptidyl peptidase III (DPP III) (EC 3.4.14.4), which has a HELLGH-E (residues 450-455, 508) motif as the zinc binding site, is classified as a zinc metallopeptidase. The zinc dissociation constants of the wild type, Leu(453)-deleted, and E508D mutant of DPP III at pH 7.4 were 4.5 (+/-0.7) x 10(-13), 5.8 (+/-0.7) x 10(-12), and 3.2 (+/-0.9) x 10(-10) M, respectively. The recoveries of the enzyme activities by the addition of various metal ions to apo-DPP III were also measured, and Co(2+), Ni(2+), and Cu(2+) ions completely recovered the enzyme activities as did Zn(2+). The dissociation constants of Co(2+), Ni(2+), and Cu(2+) ions for apo-DPP III at pH 7.4 were 8.2 (+/-0.9) x 10(-13), 2.7 (+/-0.3) x 10(-12), and 1.1 (+/-0.1) x 10(-14) M, respectively. The shape of the absorption spectrum of Co(2+)-DPP III was very similar to that of Co(2+)-carboxypeptidase A or Co(2+)-thermolysin, in which the Co(2+) is bound to two histidyl nitrogens, a water molecule, and a glutamate residue. The absorption spectrum of Cu(2+)-DPP III is also very similar to that of Cu(2+)-thermolysin. The EPR spectrum and the EPR parameters of Cu(2+)-DPP III were very similar to those of Cu(2+)-thermolysin but slightly different from those of Cu(2+)-carboxypeptidase A. The five lines of the superfine structure in the perpendicular region of the EPR spectrum in Cu(2+)-DPP III suggest that nitrogen atoms should coordinate to the cupric ion in Cu(2+)-DPP III. All of these data suggest that the donor set and the coordination geometry of the metal ions in DPP III, which has the HExxxH motif as the metal binding site, are very similar to those of the metal ions in thermolysin, which has the HExxH motif.     

Effect of sequences of the active-site dipeptides of DsbA and DsbC on in vivo folding of multidisulfide proteins in Escherichia coli

We have examined the role of the active-site CXXC central dipeptides of DsbA and DsbC in disulfide bond formation and isomerization in the Escherichia coli periplasm. DsbA active-site mutants with a wide range of redox potentials were expressed either from the trc promoter on a multicopy plasmid or from the endogenous dsbA promoter by integration of the respective alleles into the bacterial chromosome. The dsbA alleles gave significant differences in the yield of active murine urokinase, a protein containing 12 disulfides, including some that significantly enhanced urokinase expression over that allowed by wild-type DsbA. No direct correlation between the in vitro redox potential of dsbA variants and the urokinase yield was observed. These results suggest that the active-site CXXC motif of DsbA can play an important role in determining the folding of multidisulfide proteins, in a way that is independent from DsbA's redox potential. However, under aerobic conditions, there was no significant difference among the DsbA mutants with respect to phenotypes depending on the oxidation of proteins with few disulfide bonds. The effect of active-site mutations in the CXXC motif of DsbC on disulfide isomerization in vivo was also examined. A library of DsbC expression plasmids with the active-site dipeptide randomized was screened for mutants that have increased disulfide isomerization activity. A number of DsbC mutants that showed enhanced expression of a variant of human tissue plasminogen activator as well as mouse urokinase were obtained. These DsbC mutants overwhelmingly contained an aromatic residue at the C-terminal position of the dipeptide, whereas the N-terminal residue was more diverse. Collectively, these data indicate that the active sites of the soluble thiol- disulfide oxidoreductases can be modulated to enhance disulfide isomerization and protein folding in the bacterial periplasmic space.     

Substrate-assisted cysteine deprotonation in the mechanism of dimethylargininase (DDAH) from Pseudomonas aeruginosa

The enzyme dimethylargininase (also known as dimethylarginine dimethylaminohydrolase or DDAH; EC 3.5.3.18) catalyzes the hydrolysis of endogenous nitric oxide synthase inhibitors, N(omega)-methyl-l-arginine and N(omega),N(omega)-dimethyl-l-arginine. Understanding the mechanism and regulation of DDAH activity is important for developing ways to control nitric oxide production during angiogenesis and in many cases of vascular endothelial pathobiology. Several possible physiological regulation mechanisms of DDAH depend upon the presence of an active-site cysteine residue, Cys249 in Pseudomonas aeruginosa (Pa) DDAH, which is proposed to serve as a nucleophile in the catalytic mechanism. Through the use of pH-dependent ultraviolet and visible (UV-vis) difference spectroscopy and inactivation kinetics, the pK(a) of the active-site Cys249 in the resting enzyme was found to be unperturbed from pK(a) values of typical noncatalytic cysteine residues. In contrast, the pH dependence of k(cat) values indicates a much lower apparent pK(a) value. UV-vis difference spectroscopy between wild-type and C249S DDAH shows absorbance changes consistent with Cys249 deprotonation to the anionic thiolate upon binding positively charged ligands. The proton from Cys249 is lost either to the solvent or to an unidentified general base. A mutation of the active-site histidine residue, H162G, does not eliminate cysteine nucleophilicity, further arguing against a pre-formed ion pair with Cys249. Finally, UV-vis and X-ray absorption spectroscopy revealed that inhibitory metal ions can bind at these two active-site residues, Cys249 and His162, and also stabilize the anionic form of Cys249. These results support a proposed substrate-assisted mechanism for Pa DDAH in which ligand binding modulates the reactivity of the active-site cysteine.     

Reconstitution studies using the helical and carboxy-terminal domains of enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system

Enzyme I of the bacterial phosphoenolpyruvate:sugar phosphotransferase system can be phosphorylated by PEP on an active-site histidine residue, localized to a cleft between an alpha-helical domain and an alpha/beta domain on the amino terminal half of the protein. The phosphoryl group on the active-site histidine can be passed to an active-site histidine residue of HPr. It has been proposed that the major interaction between enzyme I and HPr occurs via the alpha-helical domain of enzyme I. The isolated recombinant alpha-helical domain (residues 25-145) with approximately 80% alpha-helices as well as enzyme I deficient in that domain [EI(DeltaHD)] with approximately 50% alpha-helix content from M. capricolum were used to further elucidate the nature of the enzyme I-HPr complex. Isothermal titration calorimetry demonstrated that HPr binds to the alpha-helical domain and intact enzyme I with = 5 x 10(4) and 1.4 x 10(5) M(-)(1) at pH 7.5 and 25 degrees C, respectively, but not to EI(DeltaHD), which contains the active-site histidine of enzyme I and can be autophosphorylated by PEP. In vitro reconstitution experiments with proteins from both M. capricolum and E. coli showed that EI(DeltaHD) can donate its bound phosphoryl group to HPr in the presence of the isolated alpha-helical domain. Furthermore, M. capricolum recombinant C-terminal domain of enzyme I (EIC) was shown to reconstitute phosphotransfer activity with recombinant N-terminal domain (EIN) approximately 5% as efficiently as the HD-EI(DeltaHD) pair. Recombinant EIC strongly self-associates ( approximately 10(10) M(-)(1)) in comparison to dimerization constants of 10(5)-10(7) M(-)(1) measured for EI and EI(DeltaHD).     

Affinity labeling of spinach phosphoribulokinase subsequent toS-methylation at Cys16

The chloroplast enzyme phosphoribulokinase is reversibly deactivated by oxidation of Cys16 and Cys55 to a disulfide. Although not required for catalysis, Cys16 is an active-site residue positioned at the nucleotide-binding domain (Porter and Hartman, 1988). The hyperreactivity of Cys16 has heretofore limited further active-site characterization by chemical modification. To overcome this limitation, the partially active enzyme,S-methylated at Cys16, has been probed with a potential affinity reagent. Treatment of methylated enzyme with bromoacetylethanolamine phosphate results in essentially complete loss of catalytic activity. Inactivation follows pseudo-first-order kinetics and exhibits a rate saturation with an apparentK _d of 3–4 mM. ATP, but not ribulose 5-phosphate, affords substantial protection. Complete inactivation correlates with incorporation of 1 mol of [¹⁴C]reagent per mole of enzyme subunit. Amino acid analysis of the [¹⁴C]-labeled enzyme demonstrates that only cysteine is modified, and mapping of tryptic digests shows that Cys55 is a major site of alkylation. These results indicate that Cys55 is also located in the ATP-binding domain of the active-site.     

Affinity labeling of spinach phosphoribulokinase subsequent toS-methylation at Cys16

The chloroplast enzyme phosphoribulokinase is reversibly deactivated by oxidation of Cys16 and Cys55 to a disulfide. Although not required for catalysis, Cys16 is an active-site residue positioned at the nucleotide-binding domain (Porter and Hartman, 1988). The hyperreactivity of Cys16 has heretofore limited further active-site characterization by chemical modification. To overcome this limitation, the partially active enzyme,S-methylated at Cys16, has been probed with a potential affinity reagent. Treatment of methylated enzyme with bromoacetylethanolamine phosphate results in essentially complete loss of catalytic activity. Inactivation follows pseudo-first-order kinetics and exhibits a rate saturation with an apparentK _d of 3–4 mM. ATP, but not ribulose 5-phosphate, affords substantial protection. Complete inactivation correlates with incorporation of 1 mol of [¹⁴C]reagent per mole of enzyme subunit. Amino acid analysis of the [¹⁴C]-labeled enzyme demonstrates that only cysteine is modified, and mapping of tryptic digests shows that Cys55 is a major site of alkylation. These results indicate that Cys55 is also located in the ATP-binding domain of the active-site.     

Long-range effects and conformational flexibility of aldolase

The conformational flexibility and long-range interactions in rabbit muscle aldolase induced by active-site ligand binding, cross-linking of the enzyme between Cys72 and Cys338, and removal of the C-terminal tyrosine residue were studied by following the changes in the microenvironments of Cys239 and Cys289 located outside the active site. It was found that substrates induced a conformational change in aldolase, which propagates from the active site to Cys239, which is located close to intersubunit contacts. The response of the enzyme is differential. Ligands having both C-1 and C-6 phosphates or C-1 phosphate only induce the enhancement of Cys239 reactivity, whereas those with C-6 phosphates only decrease Cys239 reactivity. This correlates well with a dramatic difference in kinetic parameters for a cleavage of fructose-1,6-P2 and fructose-1-P. Therefore, these changes can be interpreted as syncatalytic. Cross-linking of the aldolase subunit by an -S-S-bridge between Cys72 and Cys338 inactivates the enzyme, abolishes binding of active-site ligands, and induces a conformational change in the enzyme that can be detected far away (at Cys239 and Cys289) from the site of perturbation. Cys72 and Cys338 are not in the active site. This shows that the region of the active site and the environment of Cys72 and Cys338 are tightly coupled and that residues far away from the active site, through such coupling, can possess properties of active-site residues. Similar, although less dramatic changes are observed upon removal of the C-terminal tyrosine residue. In view of the results obtained in this paper, aldolase seems to be quite a flexible molecule, whose conformation is sensitive to the nature of a substrate bound to the enzyme and is able to transmit the information about a local perturbation over long distances within a molecule.     

Properties of Cys21-mutated muscle acylphosphatases

Cys21 is an invariant residue in muscle acylphosphatases, but is absent in the erythrocyte isozymes. To assess the importance of this residue in the muscle isozymes for catalytic, structural, and stability properties, two gene mutants have been prepared by oligonucleotide-directed mutagenesis and expressed inEscherichia coli cells; in these mutants, the codon for Cys21 was replaced by those for Ser and Ala, respectively. The two mutant enzymes, purified by immunoaffinity chromatography, showed kinetic and structural properties similar to those of the wild-type recombinant enzyme; however, the specific activity of the two mutants, especially that of the C21A mutant, was lower. The urea and thermal stabilities of the mutant enzymes were reduced with respect to those of the wild-type form, contrary to the susceptibility to inactivation by mercuric ions. The reported data support the possibility that Cys21 is involved in the stabilization of the enzyme active-site conformation.     

Properties of the Escherichia coli rhodanese-like protein SseA: contribution of the active-site residue Ser240 to sulfur donor recognition.

The product of Escherichia coli sseA gene (SseA) was the subject of the present investigation aimed to provide a tool for functional classification of the bacterial proteins of the rhodanese family. E. coli SseA contains the motif CGSGVTA around the catalytic cysteine (Cys238). In eukaryotic sulfurtransferases this motif discriminates for 3-mercaptopyruvate:cyanide sulfurtransferase over thiosulfate:cyanide sulfurtransferases (rhodanese). The biochemical characterization of E. coli SseA allowed the identification of the first prokaryotic protein with a preference for 3-mercaptopyruvate as donor substrate. Replacement of Ser240 with Ala showed that the presence of a hydrophobic residue did not affect the binding of 3-mercaptopyruvate, but strongly prevented thiosulfate binding. On the contrary, substitution of Ser240 with an ionizable residue (Lys) increased the affinity for thiosulfate.     

In rat dipeptidyl peptidase III,His is essential for catalysis,and Glu or Glu stabilizes the coordination bond between His or His and zinc ion

Dipeptidyl peptidase (DPP) III is a zinc-dependent exopeptidase that has a unique motif, “HELLGH,” as the zinc-binding site. In the present study, a three-dimensional (3D) model of rat DPP III was generated with the X-ray crystal structure of human DPP III (PDB: 3FVY [Dobrovetsky E. et al. (2009) SGC]) as a template. The replacement of the seven charged amino acid residues with a hydrophobic amino acid around the zinc ion did not cause any significant changes in K_m values or in the substrate specificity. However, the k_cat values of H568R and H568Y were remarkably reduced, by factors of 50 and 400, respectively. The His⁵⁶⁸ residue of rat DPP III is essential for enzyme catalysis. The k_cat values of the mutants E507A and E512A were 2.38 and 3.88 s^− 1 toward Arg-Arg-NA, and 0.097 and 0.59 s⁻¹ toward Phe-Arg-NA, respectively. These values were markedly lower than those of the wild-type DPP III. Furthermore, the zinc contents of E507A and E512A were 0.29 and 0.08 atom per mol of protein, respectively, and those mutations caused remarkable increases in the dissociation constants of the zinc ions from DPP III by factors of 5 × 10³ to 2 × 10⁴. The 3D model of the catalytic domain of rat DPP III showed that the carboxyl oxygen atoms of Glu⁵⁰⁷ and Glu⁵¹² form the hydrogen bonds to the nitrogen atoms of His⁴⁵⁵ and His⁴⁵⁰. All of these results showed that Glu⁵⁰⁷ or Glu⁵¹² stabilizes the coordination bond between the zinc ion and His⁴⁵⁵ or His⁴⁵⁰.