The binding mechanism of the yeast F1-ATPase inhibitory peptide: role of catalytic intermediates and enzyme turnover

The mechanism of inhibition of yeast mitochondrial F(1)-ATPase by its natural regulatory peptide, IF1, was investigated by correlating the rate of inhibition by IF1 with the nucleotide occupancy of the catalytic sites. Nucleotide occupancy of the catalytic sites was probed by fluorescence quenching of a tryptophan, which was engineered in the catalytic site (beta-Y345W). Fluorescence quenching of a beta-Trp(345) indicates that the binding of MgADP to F(1) can be described as 3 binding sites with dissociation constants of K(d)(1) = 10 +/- 2 nm, K(d2) = 0.22 +/- 0.03 microm, and K(d3) = 16.3 +/- 0.2 microm. In addition, the ATPase activity of the beta-Trp(345) enzyme followed simple Michaelis-Menten kinetics with a corresponding K(m) of 55 microm. Values for the K(d) for MgATP were estimated and indicate that the K(m) (55 microm) for ATP hydrolysis corresponds to filling the third catalytic site on F(1). IF1 binds very slowly to F(1)-ATPase depleted of nucleotides and under unisite conditions. The rate of inhibition by IF1 increased with increasing concentration of MgATP to about 50 mum, but decreased thereafter. The rate of inhibition was half-maximal at 5 microm MgATP, which is 10-fold lower than the K(m) for ATPase. The variations of the rate of IF1 binding are related to changes in the conformation of the IF1 binding site during the catalytic reaction cycle of ATP hydrolysis. A model is proposed that suggests that IF1 binds rapidly, but loosely to F(1) with two or three catalytic sites filled, and is then locked in the enzyme during catalytic hydrolysis of ATP.     

Platyhelminth mitochondrial and cytosolic redox homeostasis is controlled by a single thioredoxin glutathione reductase and dependent on selenium and glutathione

Platyhelminth parasites are a major health problem in developing countries. In contrast to their mammalian hosts, platyhelminth thiol-disulfide redox homeostasis relies on linked thioredoxin-glutathione systems, which are fully dependent on thioredoxin-glutathione reductase (TGR), a promising drug target. TGR is a homodimeric enzyme comprising a glutaredoxin domain and thioredoxin reductase (TR) domains with a C-terminal redox center containing selenocysteine (Sec). In this study, we demonstrate the existence of functional linked thioredoxin-glutathione systems in the cytosolic and mitochondrial compartments of Echinococcus granulosus, the platyhelminth responsible for hydatid disease. The glutathione reductase (GR) activity of TGR exhibited hysteretic behavior regulated by the [GSSG]/[GSH] ratio. This behavior was associated with glutathionylation by GSSG and abolished by deglutathionylation. The K(m) and k(cat) values for mitochondrial and cytosolic thioredoxins (9.5 microm and 131 s(-1), 34 microm and 197 s(-1), respectively) were higher than those reported for mammalian TRs. Analysis of TGR mutants revealed that the glutaredoxin domain is required for the GR activity but did not affect the TR activity. In contrast, both GR and TR activities were dependent on the Sec-containing redox center. The activity loss caused by the Sec-to-Cys mutation could be partially compensated by a Cys-to-Sec mutation of the neighboring residue, indicating that Sec can support catalysis at this alternative position. Consistent with the essential role of TGR in redox control, 2.5 microm auranofin, a known TGR inhibitor, killed larval worms in vitro. These studies establish the selenium- and glutathione-dependent regulation of cytosolic and mitochondrial redox homeostasis through a single TGR enzyme in platyhelminths.     

Discovery of a novel covalent non-β-lactam inhibitor of the metallo-β-lactamase NDM-1

The inhibition of metallo-β-lactamases (MBL) can prevent the hydrolysis of β-lactam antibiotics and hence is a promising strategy for the treatment of antibiotic resistant infections. In this study, we present a novel reversible covalent inhibitor of the clinically relevant MBL New Delhi metallo-β-lactamase 1 (NDM-1). Electrospray ionization-mass spectrometry (ESI-MS) and single site directed mutagenesis were used to show that the inhibitor forms a covalent bond with Lys224 in the active site of NDM-1. The inhibitor was further characterized using an enzyme inhibition assay, a surface plasmon resonance (SPR) based biosensor assay and covalent docking. The determined inhibition constant (K_I¹) was 580 nM and the inhibition constant for the initial complex (K_I) was 76 μM. To our knowledge, this inhibitor is the first example for a reversible covalent non-β-lactam inhibitor targeting NDM-1 and a promising starting point for the design of potent covalent inhibitors.     

Photoaffinity labeling of the aglycon binding site of the recombinant human liver UDP-glucuronosyltransferase UGT1A6 with 7-azido-4-methylcoumarin.

7-Azido-4-methylcoumarin (AzMC) is a fluorescent photoactive compound structurally related to 4-methylumbelliferone (4-MU), a marker substrate of the human liver recombinant UDP-glucuronosyltransferase (UGT) 1A6. AzMC was synthesized and utilized to label the substrate binding site of UGT1A6. AzMC exhibits a fluorescence spectrum with maximum excitation and emission wavelengths of 380 and 442 nm, respectively. Upon irradiation, the probe irreversibly inhibited glucuronidation activity measured with para-nitrophenol (pNP) as substrate and interacted with UGT1A6 according to a saturable process indicative of reversible binding before covalent incorporation of the photoaffinity label. This inhibition was both time and concentration dependent and led to the calculation of an inhibition constant, k(2) = 0.113 mM min(-1), and dissociation constant, K(d) = 2.89 mM, for the reaction. Partial photoinactivation of UGT1A6 with AzMC revealed that the probe decreased the apparent V(max) of the pNP glucuronidation reaction, but not the K(m). Moreover, inhibition was partially prevented by 1-naphthol, a surrogate substrate for the enzyme, or by preincubation with an active-site directed inhibitor, 5'-O-[[(2-decanoylamino-3-phenyl-propyloxycarbonyl)amino]-su lfonyl]-2 ',3'-O-isopropylideneuridine. In contrast, UDP-glucuronic acid (UDP-GlcUA) did not have any protective effect against photoinactivation and AzMC did not affect the photoaffinity labeling of UGT1A6 by 5-[beta-(32)P]N(3)UDP-GlcUA, a photoaffinity analog of UDP-GlcUA. Additionally, in the absence of irradiation, AzMC was found to be a competitive inhibitor of 4MU glucuronidation. Collectively, these results strongly indicate that AzMC specifically binds to the UGT1A6 aglycon binding site. Amino acid alignment of phenol-binding proteins revealed a conserved motif, YXXXKXXPXP. It is possible that this motif is involved in phenol binding to UGT1A6 and other phenol-accepting proteins.     

Species-specific irreversible inhibition of Neisseria gonorrhoeae dihydrofolate reductase by a substituted 2,4-diamino-5-benzylpyrimidine

Neisseria gonorrhoeae dihydrofolate reductase undergoes a time-dependent, irreversible inactivation by 2,4-diamino-5-[3,5-dimethoxy-4-(p-bromoacetamidophenoxy)benzyl] pyrimidine. The kinetics of inactivation are consistent with the reversible formation of an enzyme-inhibitor complex followed by covalent binding to the enzyme. The reversible component is competitive with dihydrofolate and has an inhibitor binding constant of 10 nM. Irreversible inactivation proceeds as a pseudo first-order process with a minimum inactivation half-time of 20 min and a Ki of 28 nM. Using radiolabeled inhibitor, it was shown that approximately 1 mol of ligand was covalently bound to the enzyme/mol of methotrexate binding site when the enzyme was completely inhibited. Radiolabeled inhibitor remained associated with the enzyme following denaturation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cyanogen bromide cleavage of the 14C-labeled enzyme-inhibitor complex yielded only one radioactive polypeptide, and sequence determinations showed that His-25 was modified by covalent attachment of the inhibitor. When dihydrofolate reductases from Lactobacillus casei, Streptococcus faecium, Escherichia coli, SR-1 rodent lymphoma, and chicken liver were tested with the affinity label, only the L. casei enzyme showed a time-dependent increase in inhibition. These data, along with comparisons of known amino acid sequences and x-ray crystal structures, were used to make predictions concerning the three-dimensional conformation of the gonococcal enzyme.     

Slow-tight binding inhibition of xylanase by an aspartic protease inhibitor: kinetic parameters and conformational changes that determine the affinity and selectivity of the bifunctional nature of the inhibitor

The first report of slow-tight inhibition of xylanase by a bifunctional inhibitor alkalo-thermophilic Bacillus inhibitor (ATBI), from an extremophilic Bacillus sp. is described. ATBI inhibits aspartic protease (Dash, C., and Rao, M. (2001) J. Biol. Chem., 276, 2487-2493) and xylanase (Xyl I) from a Thermomonospora sp. The steady-state kinetics revealed time-dependent competitive inhibition of Xyl I by ATBI, consistent with two-step inhibition mechanism. The inhibition followed a rapid equilibrium step to form a reversible enzyme-inhibitor complex (EI), which isomerizes to the second enzyme-inhibitor complex (EI*), which dissociated at a very slow rate. The rate constants determined for the isomerization of EI to EI*, and the dissociation of EI* were 13 +/- 1 x 10(-6) s(-1) and 5 +/- 0.5 x 10(-8) s(-1), respectively. The K(i) value for the formation of EI complex was 2.5 +/- 0.5 microm, whereas the overall inhibition constant K(i)* was 7 +/- 1 nm. The conformational changes induced in Xyl I by ATBI were monitored by fluorescence spectroscopy and the rate constants derived were in agreement with the kinetic data. Thus, the conformational alterations were correlated to the isomerization of EI to EI*. ATBI binds to the active site of the enzyme and disturbs the native interaction between the histidine and lysine, as demonstrated by the abolished isoindole fluorescence of o-phthalaldehyde (OPTA)-labeled Xyl I. Our results revealed that the inactivation of Xyl I is due to the disruption of the hydrogen-bonding network between the essential histidine and other residues involved in catalysis and a model depicting the probable interaction between ATBI or OPTA with Xyl I has been proposed.     

Interaction of a glutathione S-conjugate with glutathione reductase. Kinetic and X-ray crystallographic studies

S-Conjugates of glutathione influence the glutathione/glutathione disulfide (GSH/GSSG) status of hepatocytes in at least two ways, namely by inhibition of GSSG transport into the bile [Akerboom et al. (1982) FEBS Lett. 140, 73-76] and by inhibition of the enzyme GSSG reductase (EC 1.6.4.2). The interaction of GSSG reductase with a well-studied conjugate, namely S-(2,4-dinitrophenyl)-glutathione and its electrophilic precursor 1-chloro-2,4-dinitrobenzene are described. For short exposures both compounds are reversible inhibitors of the enzyme, the Ki values being 30 microM and 22 microM respectively. After prolonged incubation, 1-chloro-2,4-dinitrobenzene blocks GSSG reductase irreversibly, which emphasizes the need for rapid conjugate formation in situ. As shown by X-ray crystallography the major binding site of S-(2,4-dinitrophenyl)-glutathione in GSSG reductase overlaps the binding site of the substrate, glutathione disulfide. However, the glutathione moiety of the conjugate does not bind in the same manner as either of the glutathiones in the disulfide.     

Unusual inhibition of monoamine oxidase activity induced by clorgyline

The phenomenology of inhibition of FAD-containing type A monoamine oxidase by clorgyline solutions containing negligibly small amounts of clorgyline that are insufficient for stoichiometric covalent blocking of a perceptible amount of the coenzyme was studied. The nature of this phenomenon consists in the fact that at monoamine oxidase concentrations of about 10(-8) M, more than 50% of the enzyme activity in inhibited by clorgyline (less than or equal to 10(-10) M), although is accordance with a well-defined mechanism after monoamine oxidase-catalyzed tautomerization clorgyline presumably interacts with FAD at a 1:1 stoichiometric ratio. This effect termed as secondary inhibition seems to be induced not by clorgyline proper, not by changes in the solvent induced by this compound. In other words, clorgyline may initiate the synthesis of a new hypothetical inhibitor (IIC) in aqueous media which causes a reversible inhibition of the same specific inhibitory site of the enzyme. This site is responsible for the initial binding of acetylene inhibitors and catalyzes the formation of their allenic derivatives.     

Truncated mutants of human thioredoxin reductase 1 do not exhibit glutathione reductase activity

The substrate spectrum of human thioredoxin reductase (hTrxR) is attributed to its C-terminal extension of 16 amino acids carrying a selenocysteine residue. The concept of an evolutionary link between thioredoxin reductase and glutathione reductase (GR) is presently discussed and supported by the fact that almost all residues at catalytic and substrate recognition sites are identical. Here, we addressed the question if a deletion of the C-terminal part of TrxR leads to recognition of glutathione disulfide (GSSG), the substrate of GR. We introduced mutations at the putative substrate binding site to enhance GSSG binding and turnover. However, none of these enzyme species accepted GSSG as substrate better than the full length cysteine mutant of TrxR, excluding a role of the C-terminal extension in preventing GSSG binding. Furthermore, we show that GSSG binding at the N-terminal active site of TrxR is electrostatically disfavoured.     

Inhibition of CYP2B4 by the mechanism-based inhibitor 2-ethynylnaphthalene: inhibitory potential of 2EN is dependent on the size of the substrate

2-Ethynylnaphthalene (2EN) is a mechanism-based inhibitor of CYP2B4 with two components to the inhibition, (1) enzyme inactivation, which requires covalent binding of the 2EN metabolite, and (2) reversible inhibition by 2EN itself. Both inhibitory components were examined using several different CYP2B4 substrates. Preincubation of CYP2B4 with 2EN led to a time-dependent inactivation of each of the CYP2B4-dependent activities examined; however, the ability of 2EN to reversibly inhibit CYP2B4 depended on the substrate employed, which is inconsistent with classical inhibition patterns. The degree 2EN's reversible inhibition was shown not to correlate with the substrate affinity for the active site, but with parameters related to the molecular size of the substrate. The results are consistent with 2EN and the smaller substrates simultaneously fitting in the CYP2B4 active site, leading to very little inhibition. Larger substrates exhibited greater degrees of inhibition because of their inability to co-bind with inhibitor in the active site.     

Effects of glutathione reductase inhibition on cellular thiol redox state and related systems

Although inhibition of glutathione reductase (GR) has been demonstrated to cause a decrease in reduced glutathione (GSH) and increase in glutathione disulfide (GSSG), a systematic study of the effects of GR inhibition on thiol redox state and related systems has not been noted. By employing a monkey kidney cell line as the cell model and 2-acetylamino-3-[4-(2-acetylamino-2-carboxy-ethylsulfanylthio carbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) as a GR inhibitor, an investigation of the effects of GR inhibition on cellular thiol redox state and related systems was conducted. Our study demonstrated that, in addition to a decrease in GSH and increase in GSSG, 2-AAPA increased the ratios of NADH/NAD⁺ and NADPH/NADP⁺. Significant protein glutathionylation was observed. However, the inhibition did not affect the formation of reactive oxygen species or expression of antioxidant defense enzyme systems [GR, glutathione peroxidase, catalase, and superoxide dismutase] and enzymes involved in GSH biosynthesis [γ-glutamylcysteine synthetase and glutathione synthetase].     

Mechanistic and structural insights into the in vitro inhibitory action of hypericin on glutathione reductase purified from baker's yeast

This work aims at studying the interaction between glutathione reductase (GR) and hypericin. The type of inhibition was determined by measuring changes in GR activity at increasing concentrations of hypericin as well as at varying concentrations of glutathione disulfide (GSSG) and nicotinamide adenine dinucleotide phosphate (NADPH), and the binding pose of hypericin was predicted by molecular docking. Accordingly, hypericin emerges as an effective inhibitor of GR. When the variable substrate is GSSG, the type of inhibition is competitive. When the variable substrate is NADPH, however, the type of inhibition appears to be linear mixed‐type competitive. Our computational analyses suggest that hypericin binds in the large intermonomer cavity of GR, and that it may interfere with the normal positioning/functioning of the redox‐active disulfide center at the enzyme's active site. Overall, besides its contributory role in promoting oxidative stress via the formation of reactive oxygen species in photodynamic therapy, hypericin can also weaken cancer cells through inhibiting GR.     

Comparative in vitro effects of some metal ions on bovine kidney cortex glutathione reductase

Heavy metal pollution can arise from many sources and damage many organisms. Exposure to the metal ions can leads to a reduction in cellular antioxidant enzyme activities and lowers cellular defense against oxidative stress. In this study we have tested effects of the some metal ions on the purified bovine kidney cortex glutathione reductase (GR). Cadmium (Cd2+), nickel (Ni2+), and zinc (Zn2+) showed inhibitory effect on the enzyme. The obtained IC?? values of Cd2+, Ni2+, and Zn2+ are 0.027, 0.8, and 1 mM, respectively. Kinetic characterization of the inhibition is also investigated. Cd2+ inhibition is noncompetitive with respect to both oxidized glutathione (GSSG) (Ki(GSSG) 0.060 ± 0.005 mM) and NADPH (Ki(NADPH) 0.025 ± 0.002 mM). Ni2+ inhibition is noncompetitive with respect to GSSG (Ki(GSSG) 0.329 ± 0.016 mM) and uncompetitive with respect to NADPH (Ki(NADPH) 0.712 ± 0.047 mM). The effect of Zn2+ on GR activity is consistent with noncompetitive inhibition pattern when the varied substrate is the GSSG (Ki(GSSG) 0.091 ± 0.005 mM) and the NADPH (Ki(NADPH) 0.226 ± 0.01 mM), respectively. GR inhibition studies may be useful for understanding the mechanisms for oxidative damage associated with heavy metal toxicity.     

Nematode anticoagulant protein c2 reveals a site on factor Xa that is important for macromolecular substrate binding to human prothrombinase

The binding of recombinant nematode anticoagulant protein c2 (NAPc2) to either factor X or Xa is a requisite step in the pathway for the potent inhibition of VIIa tissue factor. We have used NAPc2 as a tight binding probe of human Xa to investigate protein substrate recognition by the human prothrombinase complex. NAPc2 binds with high affinity (K(d) approximately 1 nm) to both X and Xa in a way that does not require or occlude the active site of the enzyme. In contrast, NAPc2 is a tight binding, competitive inhibitor of protein substrate cleavage by human Xa incorporated into prothrombinase with saturating concentrations of membranes and Va. By fluorescence binding studies we show that NAPc2 does not interfere with the assembly of human prothrombinase. These are properties expected of an inhibitor that blocks protein substrate recognition by targeting extended macromolecular recognition sites (exosites) on the enzyme complex. A weaker interaction (K(d) = 260-500 nm) observed between NAPc2 and bovine X was restored to a high affinity one in a recombinant chimeric bovine X derivative containing 25 residues from the COOH terminus of the proteinase domain of human X. This region implicated in binding NAPc2 is spatially adjacent to a site previously identified as a potential exosite. Despite the weaker interaction with bovine Xa, NAPc2 was a tight binding competitive inhibitor of protein substrate cleavage by bovine prothrombinase as well. Extended enzymic surfaces elucidated with exosite-directed probes, such as NAPc2, may define a unique region of factor Xa that is modulated following its assembly into prothrombinase and in turn determines the binding specificity of the enzyme complex for its protein substrate.     

Inhibition and derivatization of the renal Na,K-ATPase by dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate

Treatment of purified renal Na,K-ATPase with dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate (H2DIDS) produces both reversible and irreversible inhibition of the enzyme activity. The reversible inhibition is unaffected by the presence of saturating concentrations of the sodium pump ligands Na+,K+, Mg2+, and ATP, while the inactivation is prevented by either ATP or K+. The kinetics of protection against inactivation indicate that K+ binds to two sites on the enzyme with very different affinities. Na+ ions with high affinity facilitate the inactivation by H2DIDS and prevent the protective effect of K+ ions. The H2DIDS-inactivated enzyme no longer exhibits a high-affinity nucleotide binding site, and the covalent binding of fluorescein isothiocyanate is also greatly reduced, but phosphorylation by Pi is unaffected. The kinetics of inactivation by H2DIDS were first order with respect to time and H2DIDS concentration. The enzyme is completely inactivated by the covalent binding of one H2DIDS molecule at pH 9 per enzyme phosphorylation site, or two H2DIDS molecules at pH 7.2. H2DIDS binds exclusively to the alpha-subunit of the Na,K-ATPase, locking the enzyme in an E2-like conformation. The profile of radioactivity, following trypsinolysis and SDS-PAGE, showed H2DIDS attachment to a 52-kDa fragment which also contains the ATP binding site. These results suggest that H2DIDS treatment modifies a specific conformationally sensitive amino acid residue on the alpha-subunit of the Na,K-ATPase, resulting in the loss of nucleotide binding and enzymatic activity.     

Azidonaphthoyl-ADP: a specific photolabel for the high-affinity nucleotide-binding sites of F1-ATPase

3'-O-[5-azidonaphthoyl]-ADP has been synthesized as a photoreactive analog to 3'-O-naphthoyl(1)-ADP which is known to bind to the high-affinity nucleotide sites of mitochondrial F1-ATPase, considered to be the catalytic sites. The photolabel in the dark acts as a ligand to F1-ATPase and as a competitive inhibitor with Ki = 11 microM. Binding to the enzyme is accompanied by a quench of endogenous protein fluorescence leveling off at an occupancy of 1 mol/mol F1, whereas the total number of reversible sites accessible to the analog is 3 mol/mol F1 as measured by isotope studies. Covalent insertion by near ultraviolet activation of the probe yields labeling of both alpha and beta polypeptides of F1; it is accompanied by corresponding removal of reversible high-affinity sites for ADP or naphthoyl-ADP and by an inhibition of the enzyme; total inactivation occurs at a covalent occupancy of 2 mol/mol F1. This is the maximum number of sites accessible to covalent modification by the label; one reversible site is still available in the totally inactivated enzyme. This observation is discussed in terms of a stochastic model requiring a minimum of two interacting catalytic domains out of three in order to commence catalysis.     

Structural analysis of flavinylation in vanillyl-alcohol oxidase

Vanillyl-alcohol oxidase (VAO) is member of a newly recognized flavoprotein family of structurally related oxidoreductases. The enzyme contains a covalently linked FAD cofactor. To study the mechanism of flavinylation we have created a design point mutation (His-61 --> Thr). In the mutant enzyme the covalent His-C8alpha-flavin linkage is not formed, while the enzyme is still able to bind FAD and perform catalysis. The H61T mutant displays a similar affinity for FAD and ADP (K(d) = 1.8 and 2.1 microm, respectively) but does not interact with FMN. H61T is about 10-fold less active with 4-(methoxymethyl)phenol) (k(cat) = 0.24 s(-)(1), K(m) = 40 microm) than the wild-type enzyme. The crystal structures of both the holo and apo form of H61T are highly similar to the structure of wild-type VAO, indicating that binding of FAD to the apoprotein does not require major structural rearrangements. These results show that covalent flavinylation is an autocatalytical process in which His-61 plays a crucial role by activating His-422. Furthermore, our studies clearly demonstrate that in VAO, the FAD binds via a typical lock-and-key approach to a preorganized binding site.     

Mechanism of Gly-Pro-pNA cleavage catalyzed by dipeptidyl peptidase-IV and its inhibition by saxagliptin (BMS-477118)

Dipeptidyl peptidase-IV (DPP-IV) is a serine protease with a signature Asp-His-Ser motif at the active site. Our pH data suggest that Gly-Pro-pNA cleavage catalyzed by DPP-IV is facilitated by an ionization of a residue with a pK of 7.2 +/- 0.1. By analogy to other serine proteases this pK is suggestive of His-Asp assisted Ser addition to the P1 carbonyl carbon of the substrate to form a tetrahedral intermediate. Solvent kinetic isotope effect studies yielded a D2Okcat/Km=2.9+/-0.2 and a D2Okcat=1.7+/-0.2 suggesting that kinetically significant proton transfers contribute to rate limitation during acyl intermediate formation (leaving group release) and hydrolysis. A "burst" of product release during pre steady-state Gly-Pro-pNA cleavage indicated rate limitation in the deacylation half-reaction. Nevertheless, the amplitude of the burst exceeded the enzyme concentration significantly (approximately 15-fold), which is consistent with a branching deacylation step. All of these data allowed us to better understand DPP-IV inhibition by saxagliptin (BMS-477118). We propose a two-step inhibition mechanism wherein an initial encounter complex is followed by covalent intermediate formation. Final inhibitory complex assembly (kon) depends upon the ionization of an enzyme residue with a pK of 6.2 +/- 0.1, and we assigned it to the catalytic His-Asp pair which enhances Ser nucleophilicity for covalent addition. An ionization with a pK of 7.9 +/- 0.2 likely reflects the P2 terminal amine of the inhibitor hydrogen bonding to Glu205/Glu206 in the enzyme active site. The formation of the covalent enzyme-inhibitor complex was reversible and dissociated with a koff of (5.5 +/- 0.4) x 10(-5) s(-1), thus yielding a Ki* (as koff/kon) of 0.35 nM, which is in good agreement with the value of 0.6 nM obtained from steady-state inhibition studies. Proton NMR spectra of DPP-IV showed a downfield resonance at 16.1 ppm. Two additional peaks in the 1H NMR spectra at 17.4 and 14.1 ppm were observed upon mixing the enzyme with saxagliptin. Fractionation factors (phi) of 0.6 and 0.5 for the 17.4 and 14.1 ppm peaks, respectively, are suggestive of short strong hydrogen bonds in the enzyme-inhibitor complex.