Role of a propeller loop in the quaternary structure and enzymatic activity of prolyl dipeptidases DPP-IV and DPP9

The dipeptidyl peptidase (DPP) family members, including DPP-IV, DPP8, DPP9 and others, cleave the peptide bond after the penultimate proline residue and are drug target rich. The dimerization of DPP-IV is required for its activity. A propeller loop located at the dimer interface is highly conserved within the family. Here we carried out site-directed mutagenesis on the loop of DPPIV and identified several residues important for dimer formation and enzymatic activity. Interestingly, the corresponding residues on DPP9 have a different impact whereby the mutations decrease activity without changing dimerization. Thus the propeller loop seems to play a varying role in different DPPs.     

One site mutation disrupts dimer formation in human DPP-IV proteins

DPP-IV is a prolyl dipeptidase, cleaving the peptide bond after the penultimate proline residue. It is an important drug target for the treatment of type II diabetes. DPP-IV is active as a dimer, and monomeric DPP-IV has been speculated to be inactive. In this study, we have identified the C-terminal loop of DPP-IV, highly conserved among prolyl dipeptidases, as essential for dimer formation and optimal catalysis. The conserved residue His750 on the loop contributes significantly for dimer stability. We have determined the quaternary structures of the wild type, H750A, and H750E mutant enzymes by several independent methods including chemical cross-linking, gel electrophoresis, size exclusion chromatography, and analytical ultracentrifugation. Wild-type DPP-IV exists as dimers both in the intact cell and in vitro after purification from human semen or insect cells. The H750A mutation results in a mixture of DPP-IV dimer and monomer. H750A dimer has the same kinetic constants as those of the wild type, whereas the H750A monomer has a 60-fold decrease in kcat. Replacement of His750 with a negatively charged Glu (H750E) results in nearly exclusive monomers with a 300-fold decrease in catalytic activity. Interestingly, there is no dynamic equilibrium between the dimer and the monomer for all forms of DPP-IVs studied here. This is the first study of the function of the C-terminal loop as well as monomeric mutant DPP-IVs with respect to their enzymatic activities. The study has important implications for the discovery of drugs targeted to the dimer interface.     

Novel isoindoline compounds for potent and selective inhibition of prolyl dipeptidase DPP8

DPP8 is a prolyl dipeptidase homologous to DPP-IV, which is a drug target for Type II diabetes. The biological function of DPP8 is not known. To identify potent and selective chemical compounds against DPP8, we have synthesized a series of isoquinoline and isoindoline derivatives and have tested their inhibitory activity against DPP8, DPP-IV and DPP-II. Isoindoline derivatives were found to be more potent DPP8 inhibitors than isoquinoline derivatives. Isoindoline with a 1-(4,4'-difluor-benzhydryl)-piperazine group at the P2 site was observed to be a very potent DPP8 inhibitor, having an IC(50) value of 14nM with at least a 2500-fold selectivity over either DPP-IV or DPP-II. From SAR results, we speculate that the S1 site of DPP8 may be larger than that of DPP-IV, which would allow the accommodation of larger C-terminal residues, such as isoquinoline or isoindoline.     

Dipeptidyl peptidase IV activity and/or structure homologues (DASH) and their substrates in cancer

Post-translational modification of proteins is an important regulatory event. Numerous biologically active peptides that play an essential role in cancerogenesis contain an evolutionary conserved proline residue as a proteolytic-processing regulatory element. Proline-specific proteases could therefore be viewed as important "check-points". Limited proteolysis of such peptides may lead to quantitative but, importantly, due to the change of receptor preference, also qualitative changes of their signaling potential.Dipeptidyl peptidase-IV (DPP-IV, EC 3.4.14.5, identical with CD26) was for many years believed to be a unique cell membrane protease cleaving X-Pro dipeptides from the N-terminal end of peptides and proteins. Subsequently, a number of other molecules were discovered, exhibiting various degree of structural homology and DPP-IV-like enzyme activity, capable of cleaving similar set of substrates. These comprise for example, seprase, fibroblast activation protein alpha, DPP6, DPP8, DPP9, attractin, N-acetylated-alpha-linked-acidic dipeptidases I, II and L, quiescent cell proline dipeptidase, thymus-specific serine protease and DPP IV-beta. It is tempting to speculate their potential participation on DPP-IV biological function(s). Disrupted expression and enzymatic activity of "DPP-IV activity and/or structure homologues" (DASH) might corrupt the message carried by their substrates, promoting abnormal cell behavior. Consequently, modulation of particular enzyme activity using e.g. DASH inhibitors, specific antibodies or DASH expression modification may be an attractive therapeutic concept in cancer treatment. This review summarizes recent information on the interactions between DASH members and their substrates with respect to their possible role in cancer biology.     

Length of the active-site crossover loop defines the substrate specificity of ubiquitin C-terminal hydrolases for ubiquitin chains

UCHs [Ub (ubiquitin) C-terminal hydrolases] are a family of deubiquitinating enzymes that are often thought to only remove small C-terminal peptide tails from Ub adducts. Among the four UCHs identified to date, neither UCH-L3 nor UCH-L1 can catalyse the hydrolysis of isopeptide Ub chains, but UCH-L5 can when it is present in the PA700 complex of the proteasome. In the present paper, we report that the UCH domain of UCH-L5, different from UCH-L1 and UCH-L3, by itself can process the K48-diUb (Lys48-linked di-ubiquitin) substrate by cleaving the isopeptide bond between two Ub units. The catalytic specificity of the four UCHs is dependent on the length of the active-site crossover loop. The UCH domain with a long crossover loop (usually >14 residues), such as that of UCH-L5 or BAP1 [BRCA1 (breast cancer early-onset 1)-associated protein 1], is able to cleave both small and large Ub derivatives, whereas the one with a short loop can only process small Ub derivatives. We also found that elongation of the crossover loop enables UCH-L1 to have isopeptidase activity for K48-diUb in a length-dependent manner. Thus the loop length of UCHs defines their substrate specificity for diUb chains, suggesting that the chain flexibility of the crossover loop plays an important role in determining its catalytic activity and substrate specificity for cleaving isopeptide Ub chains.     

Identification of hydrophobic residues critical for DPP-IV dimerization

The prolyl dipeptidase DPP-IV plays diverse and important roles in cellular functions. It is a membrane-bound exoprotease involved in the proteolytic cleavage of several insulin-sensing hormones. The inhibition of its enzymatic activity has been proven effective in the treatment of type II diabetes. Homodimeric DPP-IV interacts extracellularly with adenosine deaminase, and this interaction is critical for adenosine signaling and T-cell proliferation. In this study, we investigated the contribution of hydrophobic interactions to the dimerization of DPP-IV. Hydrophobic residues F713, W734, and Y735 were found to be essential for DPP-IV dimerization. Moreover, the enzymatic activity of DPP-IV was correlated with its quaternary structure. Monomeric DPP-IV had only residual activity left, ranging from 1/30 to 1/1600 of the dimeric forms. Using a surface plasmon resonance technique, we demonstrated that the affinity of these DPP-IV monomers for adenosine deaminase was not significantly altered, compared to that of dimeric DPP-IV. The study not only identifies the hydrophobic interactions critical for DPP-IV dimer formation, but also reveals no global conformational change upon the formation of monomers as determined by the protein-protein interaction (Kd) of DPP-IV with adenosine deaminase.     

Dipeptidyl peptidase-IV inhibits glioma cell growth independent of its enzymatic activity

Malignant gliomas exhibit abnormal expression of proteolytic enzymes that may participate in the uncontrolled cell proliferation and aberrant interactions with the brain extracellular matrix. The multifunctional membrane bound serine aminopeptidase dipeptidyl peptidase (DPP)-IV has been linked to the development and progression of several malignancies, possibly both through the enzymatic and nonenzymatic mechanisms. In this report we demonstrate the expression of DPP-IV and homologous proteases fibroblast activation protein, DPP8 and DPP9 in primary cell cultures derived from high-grade gliomas, and show that the DPP-IV-like enzymatic activity is negatively associated with their in vitro growth. More importantly, the DPP-IV positive subpopulation isolated from the primary cell cultures using immunomagnetic separation exhibited slower proliferation. Forced expression of the wild as well as the enzymatically inactive mutant DPP-IV in glioma cell lines resulted in their reduced growth, migration and adhesion in vitro, as well as suppressed glioma growth in an orthotopic xenotransplantation mouse model. Microarray analysis of glioma cells with forced DPP-IV expression revealed differential expression of several candidate genes not linked to the tumor suppressive effects of DPP-IV in previous studies. Gene set enrichment analysis of the differentially expressed genes showed overrepresentation of gene ontology terms associated with cell proliferation, cell adhesion and migration. In conclusion, our data show that DPP-IV may interfere with several aspects of the malignant phenotype of glioma cells in great part independent of its enzymatic activity.     

Lead optimization of [(S)-gamma-(arylamino)prolyl]thiazolidine focused on gamma-substituent: Indoline compounds as potent DPP-IV inhibitors

Dipeptidyl peptidase IV (DPP-IV) inhibitors are looked to as a potential new antidiabetic agent class. A series of [(S)-gamma-(arylamino)prolyl]thiazolidine compounds in which the electrophilic nitrile is removed are chemically stable DPP-IV inhibitors. To discover a structure for the gamma-substituent of the proline moiety more suitable for interacting with the S(2) pocket of DPP-IV, optimization focused on the gamma-substituent was carried out. The indoline compound 22e showed a DPP-IV-inhibitory activity 100-fold more potent than that of the prolylthiazolidine 10 and comparable to that of NVP-DPP728. It also displayed improved inhibitory selectivity for DPP-IV over DPP8 and DPP9 compared to compound 10. Indoline compounds such as 22e have a rigid conformation with double restriction of the aromatic moiety by proline and indoline structures to promote interaction with the binding site in the S(2) pocket of DPP-IV. The double restriction effect provides a potent inhibitory activity which compensates for the decrease in activity caused by removing the electrophilic nitrile.     

Changing the substrate specificity of creatine kinase from creatine to glycocyamine: evidence for a highly evolved active site

Eight variants of creatine kinase were created to switch the substrate specificity from creatine to glycocyamine using a rational design approach. Changes to creatine kinase involved altering several residues on the flexible loops that fold over the bound substrates including a chimeric replacement of the guanidino specificity loop from glycocyamine kinase into creatine kinase. A maximal 2,000-fold change in substrate specificity was obtained as measured by a ratio of enzymatic efficiency (k(cat)/K(M).K(d)) for creatine vs. glycocyamine. In all cases, a change in specificity was accompanied by a large drop in enzymatic efficiency. This data, combined with evidence from other studies, indicate that substrate specificity in the phosphagen kinase family is obtained by precise alignment of substrates in the active site to maximize k(cat)/K(M).K(d) as opposed to selective molecular recognition of one guanidino substrate over another. A model for the evolution of the dimeric forms of phosphagen kinases is proposed in which these enzymes radiated from a common ancestor that may have possessed a level of catalytic promiscuity. As mutational events occurred leading to greater degrees of substrate specificity, the dimeric phosphagen kinases became evolutionary separated such that the substrate specificity could not be interchanged by a small number of mutations.     

Two highly conserved glutamic acid residues in the predicted beta propeller domain of dipeptidyl peptidase IV are required for its enzyme activity

Dipeptidyl peptidase IV (DPP IV) is a member of the prolyl oligopeptidase family and modifies the biological activities of certain chemokines and neuropeptides by cleaving their N-terminal dipeptides. This paper reports the identification and possible significance of a novel conserved sequence motif Asp-Trp-(Val/Ile/Leu)-Tyr-Glu-Glu-Glu (DW(V/I/L)YEEE) in the predicted beta propeller domain of the DPP IV-like gene family. Single amino acid point mutations in this motif identified two glutamates, at positions 205 and 206, as essential for the enzyme activity of human DPP IV. This observation suggests a novel role in proteolysis for residues of DPP IV distant from the Ser-Asp-His catalytic triad.     

Mutations in the substrate binding site of thrombin-activatable fibrinolysis inhibitor (TAFI) alter its substrate specificity

Thrombin-activable fibrinolysis inhibitor (TAFI) is a zymogen that inhibits the amplification of plasmin production when converted to its active form (TAFIa). TAFI is structurally very similar to pancreatic procarboxypeptidase B. TAFI also shares high homology in zinc binding and catalytic sites with the second basic carboxypeptidase present in plasma, carboxypeptidase N. We investigated the effects of altering residues involved in substrate specificity to understand how they contribute to the enzymatic differences between TAFI and carboxypeptidase N. We expressed wild type TAFI and binding site mutants in 293 cells. Recombinant proteins were purified and characterized for their activation and enzymatic activity as well as functional activity. Although the thrombin/thrombomodulin complex activated all the mutants, carboxypeptidase B activity of the activated mutants against hippuryl-arginine was reduced. Potato carboxypeptidase inhibitor inhibited the residual activity of the mutants. The functional activity of the mutants in a plasma clot lysis assay correlated with their chromogenic activity. The effect of the mutations on other substrates depended on the particular mutation, with some of the mutants possessing more activity against hippuryl-His-leucine than wild type TAFIa. Thus mutations in residues around the substrate binding site of TAFI resulted in altered C-terminal substrate specificity.     

Kinetic rationale for selectivity toward N- and C-terminal oxygen-dependent degradation domain substrates mediated by a loop region of hypoxia-inducible factor prolyl hydroxylases

Hydroxylation of two conserved prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of the alpha-subunit of hypoxia-inducible factor (HIF) signals for its degradation via the ubiquitin-proteasome pathway. In human cells, three prolyl hydroxylases (PHDs 1-3) belonging to the Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase family catalyze prolyl hydroxylation with differing selectivity for CODD and NODD. Sequence analysis of the catalytic domains of the PHDs in the light of crystal structures for PHD2, and results for other 2OG oxygenases, suggested that either the C-terminal region or a loop linking two beta-strands (beta2 and beta3 in human PHD2) are important in determining substrate selectivity. Mutation analyses on PHD2 revealed that the beta2beta3 loop is a major determinant in conferring selectivity for CODD over NODD peptides. A chimeric PHD in which the beta2beta3 loop of PHD2 was replaced with that of PHD3 displayed an almost complete selectivity for CODD (in competition experiments), as observed for wild-type PHD3. CODD was observed to bind much more tightly to this chimeric protein than the wild type PHD2 catalytic domain.     

Characterization of an Aspergillus oryzae cysteinyl dipeptidase expressed in Escherichia coli

Cysteinyl dipeptidase from Aspergillus oryzae (CdpA) was produced in Escherichia coli and purified. The enzyme showed activity specific toward cysteine-containing dipeptides, but its substrate specificity was distinct from those of other cysteinyl dipeptidases of the M20 family. It was optimally active at pH 7-8 and stable at pH 6-9 and at up to 40 °C.     

A single substitution of the insulin receptor kinase inhibits serine autophosphorylation in vitro: evidence for an interaction between the C-terminus and the activation loop

We examined the effects of mutations of tyrosine and serine autophosphorylation sites on the dual specificity of the insulin receptor kinase (IRKD) in vitro using autophosphorylation and substrate phosphorylation and phosphopeptide mapping. For comparable studies, the recombinant kinases were overexpressed in the baculovirus system, purified, and analyzed. The phosphate incorporation into the enzymes was in the range of 3-4.5 mol/mol, and initial velocities of autophosphorylation were reduced up to 2-fold. However, the mutation Y1151F in the activation loop inhibited phosphate incorporation in the C-terminal serine residues 1275 and 1309, due to a 10-fold decrease of the initial velocity of serine autophosphorylation. Although the K(M) and V(MAX) values of this mutant were only slightly altered in substrate phosphorylation reactions using a recombinant C-terminal insulin receptor peptide (K(M): Y1151F, 9.9 +/- 0.4 microM; IRKD, 6.1 +/- 0.2 microM; V(MAX): Y1151F, 72 +/- 4 nmol min(-)(1) mg(-)(1); IRKD, 117 +/- 6 nmol min(-)(1) mg(-)(1)), diminished phosphate incorporation into serine residues of the peptide was observed. In contrast, the phosphorylation of a recombinant IRS-1 fragment, which was shown to be phosphorylated markedly on serine residues by IRKD, was not affected by any kinase mutation. These results underline that IRKD is a kinase with dual specificity. The substrate specificity toward C-terminal serine phosphorylation sites can be modified by a single amino acid substitution in the activation loop, whereas the specificity toward IRS-1 is not affected, suggesting that the C-terminus and the activation loop interact.     

Site-directed mutagenesis of human nucleoside triphosphate diphosphohydrolase 3: the importance of residues in the apyrase conserved regions

Ecto-nucleoside triphosphate diphosphohydrolase 3 (eNTPDase-3, also known as HB6 and CD39L3) is a membrane-associated ecto-apyrase. Only a few functionally significant residues have been elucidated for this enzyme, as well as for the whole family of eNTPDase enzymes. Four highly conserved regions (apyrase conserved regions, ACRs) have been identified in all the members of eNTPDase family, suggesting their importance for biological activity. In an effort to identify those amino acids important for the catalytic activity of the eNTPDase family, as well as those residues mediating substrate specificity, 11 point mutations of 7 amino acid residues in ACR1-4 of eNTPDase-3 were constructed by site-directed mutagenesis. Mutagenesis of asparagine 191 to alanine (N191A), glutamine 226 to alanine (Q226A), and arginine 67 to glycine (R67G) resulted in an increase in the rates of hydrolysis of nucleoside diphosphates relative to triphosphates. Mutagenesis of arginine 146 to proline (R146P) essentially converted the eNTPDase-3 ecto-apyrase to an ecto-ATPase (eNTPDase-2), mainly by decreasing the hydrolysis rates for nucleoside diphosphates. The Q226A mutant exhibited a change in the divalent cation requirement for nucleotidase activity relative to the wild-type and the other mutants. Mutation of glutamate 182 to aspartate (E182D) or glutamine (E182Q), and mutation of serine 224 to alanine (S224A) completely abolished enzymatic activity. We conclude that the residues corresponding to eNTPDase-3 glutamate 182 in ACR3 and serine 224 in ACR4 are essential for the enzymatic activity of eNTPDases in general, and that arginine 67, arginine 146, asparagine 191, and glutamine 226 are important for determining substrate specificity for human ecto-nucleoside triphosphate diphosphohydrolase 3.     

Crystal structure and mechanism of tripeptidyl activity of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis

The crystal structure of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis was determined. Prolyl tripeptidyl aminopeptidase consists of beta-propeller and catalytic domains, and a large cavity between the domains; this structure is similar to dipeptidyl aminopeptidase IV. A catalytic triad (Ser603, His710, and Asp678) was located in the catalytic domain; this triad was virtually identical to that of the enzymes belonging to the prolyl oligopeptidase family. The structure of an inactive S603A mutant enzyme complexed with a substrate was also determined. The pyrrolidine ring of the proline residue appeared to fit into a hydrophobic pocket composed of Tyr604, Val629, Trp632, Tyr635, Tyr639, Val680, and Val681. There were characteristic differences in the residues of the beta-propeller domain, and these differences were related to the substrate specificity of tripeptidyl activity. The N-terminal amino group was recognized by salt bridges, with two carboxyl groups of Glu205 and Glu206 from a helix in dipeptidyl aminopeptidase IV. In prolyl tripeptidyl aminopeptidase, however, the Glu205 (located in the loop) and Glu636 were found to carry out this function. The loop structure provides sufficient space to accommodate three N-terminal residues (Xaa-Xaa-Pro) of substrates. This is the first report of the structure and substrate recognition mechanism of tripeptidyl peptidase.     

The acidic C-terminal domain of protein disulfide isomerase is not critical for the enzyme subunit function or for the chaperone or disulfide isomerase activities of the polypeptide.

Protein disulfide isomerase (PDI) is a multifunctional polypeptide that acts as a subunit in the animal prolyl 4-hydroxylases and the microsomal triglyceride transfer protein, and as a chaperone that binds various peptides and assists their folding. We report here that deletion of PDI sequences corresponding to the entire C-terminal domain c, previously thought to be critical for chaperone activity, had no inhibitory effect on the assembly of recombinant prolyl 4-hydroxylase in insect cells or on the in vitro chaperone activity or disulfide isomerase activity of purified PDI. However, partially overlapping critical regions for all these functions were identified at the C-terminal end of the preceding thioredoxin-like domain a'. Point mutations introduced into this region identified several residues as critical for prolyl 4-hydroxylase assembly. Circular dichroism spectra of three mutants suggested that two of these mutations may have caused only local alterations, whereas one of them may have led to more extensive structural changes. The critical region identified here corresponds to the C-terminal alpha helix of domain a', but this is not the only critical region for any of these functions.     

Characterization of an Archaeal Cyclodextrin Glucanotransferase with a Novel C-Terminal Domain

A gene encoding a cyclodextrin glucanotransferase (CGTase) from Thermococcus kodakaraensis KOD1 (CGT(Tk)) was identified and characterized. The gene (cgt(Tk)) encoded a protein of 713 amino acid residues harboring the four conserved regions found in all members of the alpha-amylase family. However, the C-terminal domain corresponding to domain E of previously known CGTases displayed a completely distinct primary structure. In order to elucidate the catalytic function of the gene product, the recombinant enzyme was purified by anion-exchange chromatography, and its enzymatic properties were investigated. The enzyme displayed significant starch-degrading activity (750 U/mg of protein) with an optimal temperature and pH of 80 degrees C and 5.5 to 6.0, respectively. The presence of Ca(2+) enhanced the enzyme activity and elevated the optimum temperature to 85 to 90 degrees C. With the addition of Ca(2+), the enzyme showed extreme thermostability, with almost no loss of enzymatic activity after 80 min at 85 degrees C, and a half-life of 20 min at 100 degrees C. CGT(Tk) could hydrolyze soluble starch and glycogen but failed to hydrolyze pullulan. Most importantly, although CGT(Tk) harbored a unique C-terminal domain, we found that the protein also exhibited significant CGTase activity, with beta-cyclodextrin as the main product. In order to identify the involvement, if any, of the C-terminal region in the CGTase activity, we analyzed a truncated protein (CGT(Tk)DeltaC) with 23 C-terminal amino acid residues deleted. CGT(Tk)DeltaC displayed similar properties in terms of starch-binding activity, substrate specificity, and thermostability, but unexpectedly showed higher starch-degrading activity than the parental CGT(Tk). In contrast, the cyclization activity of CGT(Tk)DeltaC was abolished. The results indicate that the presence of the structurally novel C-terminal domain is essential for CGT(Tk) to properly catalyze the cyclization reaction.     

Novel prolyl tri/tetra-peptidyl aminopeptidase from Streptomyces mobaraensis: substrate specificity and enzyme gene cloning

The prolyl peptidase that removes the tetra-peptide of pro-transglutaminase was purified from Streptomyces mobaraensis mycelia. The substrate specificity of the enzyme using synthetic peptide substrates showed proline-specific activity with not only tripeptidyl peptidase activity, but also tetrapeptidyl peptidase activity. However, the enzyme had no other exo- and endo-activities. This substrate specificity is different from proline specific peptidases so far reported. The enzyme gene was cloned, based on the direct N-terminal amino acid sequence of the purified enzyme, and the entire nucleotide sequence of the coding region was determined. The deduced amino acid sequence revealed an N-terminal signal peptide sequence (33 amino acids) followed by the mature protein comprising 444 amino acid residues. This enzyme shows no remarkable homology with enzymes belonging to the prolyl oligopeptidase family, but has about 65% identity with three tripeptidyl peptidases from Streptomyces lividans, Streptomyces coelicolor, and Streptomyces avermitilis. Based on its substrate specificity, a new name, "prolyl tri/tetra-peptidyl aminopeptidase," is proposed for the enzyme.     

Charged residues on a flap-loop structure of Lactococcus lactis prolidase play critical roles in allosteric behavior and substrate inhibition

Allosteric behavior and substrate inhibition are unique characteristics of Lactococcus lactis prolidase. We hypothesized that charged residues (Asp36, His38, Glu39, and Arg40), present on one loop essential for catalysis, interact with residues in or near the active site to impart these unique characteristics. Asp36 has a predominant role in the allosteric behavior, as demonstrated through the non-allosteric behavior of the D36S mutant enzyme. In contrast, a double mutant (D36E/R293K) maintained the allostery, indicating that this aspartic acid residue interacts with Arg293, previously shown to be critical in the allostery. Substitution of His38 drastically reduced the substrate inhibition, and substrate specificity of the mutant at Asp36 or His38 showed the influence of these residues to the substrate specificity. These findings confirm the importance of the loop in the enzymatic reaction mechanism and suggest the existence of conformational changes of the loop structure between open and closed states. A variety of mutations at Glu39 and Arg40 showed that these residues influence roles of the loop in the enzyme reaction. On the basis of these results and combined with observations of molecular models of this prolidase, we concluded that Asp36 and His38 interact with the residues in the active site to generate an allosteric subsite and a pseudo-S(1)' site, which are responsible for the allosteric behavior and substrate inhibition.