Site-directed mutagenesis of the human DNA repair enzyme HAP1: identification of residues important for AP endonuclease and RNase H activity.

HAP1 protein, the major apurinic/apyrimidinic (AP) endonuclease in human cells, is a member of a homologous family of multifunctional DNA repair enzymes including the Escherichia coli exonuclease III and Drosophila Rrp1 proteins. The most extensively characterised member of this family, exonuclease III, exhibits both DNA- and RNA-specific nuclease activities. Here, we show that the RNase H activity characteristic of exonuclease III has been conserved in the human homologue, although the products resulting from RNA cleavage are dissimilar. To identify residues important for enzymatic activity, five mutant HAP1 proteins containing single amino acid substitutions were purified and analysed in vitro. The substitutions were made at sites of conserved amino acids and targeted either acidic or histidine residues because of their known participation in the active sites of hydrolytic nucleases. One of the mutant proteins (replacement of Asp-219 by alanine) showed a markedly reduced enzymatic activity, consistent with a greatly diminished capacity to bind DNA and RNA. In contrast, replacement of Asp-90, Asp-308 or Glu-96 by alanine led to a reduction in enzymatic activity without significantly compromising nucleic acid binding. Replacement of His-255 by alanine led to only a very small reduction in enzymatic activity. Our data are consistent with the presence of a single catalytic active site for the DNA- and RNA-specific nuclease activities of the HAP1 protein.     

Akazara scallop troponin C: Ca(2+)-induced conformational change and interaction with rabbit troponin subunits.

The number of specific Ca2+ bound to Akazara scallop troponin C was estimated to be 0.7 with an apparent binding constant of 5 x 10(5) M-1 (T. Ojima and K. Nishita, 1986, J. Biol. Chem. 261, 16749-16754). In the present paper, we report on the Ca(2+)-induced conformational changes in the troponin C and the interaction of the troponin C with rabbit troponin subunits. The Ca2+ binding to the troponin C caused a marked change in difference uv absorption spectra and a retardation of elution on Sephacryl S-200 gel filtration. However, its circular dichroism spectrum was hardly changed by the Ca2+ binding. These results suggest that the Ca2+ binding to the troponin C induced changes predominantly in tertiary structure rather than in secondary structure. Akazara scallop troponin C was shown to be able to bind to rabbit troponin I-Cellulofine affinity column, but the affinity was not greatly increased by Ca2+ unlike the case of rabbit troponin C. On hybridizing with rabbit troponin T and I, Akazara scallop troponin C was shown to be incapable of substituting rabbit troponin C; i.e., the hybrid troponin strongly inhibited the Mg-ATPase activity of rabbit actomyosin-tropomyosin irrespective of the presence or absence of Ca2+, thus recovering no Ca2+ sensitivity.     

Neutrophil recognition requires a Ca(2+)-induced conformational change in the lectin domain of GMP-140.

GMP-140, a receptor for myeloid cells that is expressed on surfaces of thrombin-activated platelets and endothelial cells, is a member of the selectin family of adhesion molecules that regulate leukocyte interactions with the blood vessel wall. Each selectin contains an N-terminal domain homologous to Ca(2+)-dependent lectins and mediates cell-cell contact by binding to oligosaccharide ligands in a Ca(2+)-dependent manner. The mechanisms by which Ca2+ promotes selectin-dependent cellular interactions have not been defined. We demonstrate that purified GMP-140 contains two high affinity binding sites for Ca2+ as measured by equilibrium dialysis (Kd = 22 +/- 2 microM). Occupancy of these sites by Ca2+ alters the conformation of the protein as detected by a reduction in intrinsic fluorescence emission intensity (Kd = 4.8 +/- 0.2 microM). This Ca(2+)-dependent conformational change exposes an epitope spanning residues 19-34 of the lectin domain that is recognized by a monoclonal antibody capable of blocking neutrophil adhesion to GMP-140 (half-maximal antibody binding at approximately 20 microM Ca2+). Furthermore, a synthetic peptide encoding this epitope, CQNRYTDLVAIQNKNE, inhibits neutrophil binding to GMP-140. Mg2+ also alters the conformation of the protein, but not in a manner that will support leukocyte recognition in the absence of Ca2+. There is a strong correlation between the Ca2+ levels required for neutrophil adhesion to GMP-140, for occupancy of the two Ca(2+)-binding sites, for the fluorescence-detected conformational change, and for exposure of the antibody epitope in the lectin domain. We conclude that binding of Ca2+ to high affinity sites on GMP-140 modulates the conformation of the lectin domain in a manner that is essential for leukocyte recognition.     

The Ca(2+)-induced conformational change of gelsolin is located in the carboxyl-terminal half of the molecule.

We have purified the two functionally distinct domains of gelsolin, a Ca(2+)-dependent actin binding protein, by proteolytic cleavage and characterized their size and shape in solution by dynamic light scattering. In the absence of calcium we obtained the same translational diffusion coefficient for both fragments which are of approximately equal molecular mass. The frictional ratio fo/fexp (1.33-1.39) is similar to the value as obtained for intact gelsolin (1.37) in aqueous solution (Patkowski, A., J. Seils, H. Hinssen, and T. Dorfmüller. 1990. Biopolymers. 30:427-435), indicating a similar molecular shape for the native protein as well as for the two subdomains. Upon addition of Ca2+ the translational diffusion coefficient of the carboxyl-terminal half decreased by almost 10%, while there was no change observed for the amino terminus. This result indicates that the ligand-induced conformational change as seen for intact gelsolin is probably located on the carboxyl-terminal domain of the protein. Since gelsolin has binding sites in both domains, and the isolated amino terminus binds and severs actin in a calcium-independent manner, our results suggests that the structural transition in the carboxyl-terminal part of intact gelsolin also affects the actin binding properties of the amino-terminal half.     

Purification and characterization of a Ca(2+)-dependent protein kinase from sandalwood (Santalum album L.): evidence for Ca(2+)-induced conformational changes

An early development-specific soluble 55 kDa Ca(2+)-dependent protein kinase has been purified to homogeneity from sandalwood somatic embryos and biochemically characterized. The purified enzyme, swCDPK, resolved into a single band on 10% polyacrylamide gels, both under denaturing and non-denaturing conditions. swCDPK activity was strictly dependent on Ca(2+), K(0.5) (apparent binding constant) for Ca(2+)-activation of substrate phosphorylation activity being 0.7 microM and for autophosphorylation activity approximately 50 nM. Ca(2+)-dependence for activation, CaM-independence, inhibition by CaM-antagonist (IC(50) for W7=6 microM, for W5=46 microM) and cross-reaction with polyclonal antibodies directed against the CaM-like domain of soybean CDPK, confirmed the presence of an endogenous CaM-like domain in the purified enzyme. Kinetic studies revealed a K(m) value of 1.3 mg/ml for histone III-S and a V(max) value of 0.1 nmol min(-1) mg(-1). The enzyme exhibited high specificity for ATP with a K(m) value of 10 nM. Titration with calcium resulted in the enhancement of intrinsic emission fluorescence of swCDPK and a shift in the lambda(max) emission from tryptophan residues. A reduction in the efficiency of non-radiative energy transfer from tyrosine to tryptophan residues was also observed. These are taken as evidence for the occurrence of Ca(2+)-induced conformational change in swCDPK. The emission spectral properties of swCDPK in conjunction with Ca(2+) levels required for autophosphorylation and substrate phosphorylation help understand mode of Ca(2+) activation of this enzyme.     

Ca2(+)-induced conformational change and aggregation of chromogranin A

Chromogranin A, the most abundant protein in bovine adrenal chromaffin granules, bound calmodulin in a Ca2(+)-dependent manner, and the calmodulin-binding property was utilized to purify chromogranin A. Chromogranin A has been described in the past as a "random-coil polypeptide" with little alpha-helix or beta-sheet conformation. However, circular dichroism measurements with pure, native chromogranin A revealed relatively high alpha-helical contents (40% at the intravesicular pH of 5.5). Fluorescence studies confirmed previous observations that chromogranin A binds Ca2+ with low affinity. Considering the high concentration of Ca2+ in the secretory vesicle, the effect of Ca2+ on the secondary structure and self-association of chromogranin A was examined. Ca2+ induced a decrease of alpha-helicity of chromogranin A from 40 to 30% at pH 5.5. In contrast, at pH 7.5 the same amount of Ca2+ increased alpha-helicity of the protein from 25 to 40%. Boiling of the adrenal extract, a commonly used purification procedure for chromogranin A, resulted in the isolation of conformationally distinct chromogranin A molecule. Unlike secretory protein-I of the parathyroid gland (Gorr, S.-V., Dean, W. L., Radley, T. L., and Cohn, D. V. (1988) Bone Mineral 4, 17-25), chromogranin A aggregated rapidly in the presence of Ca2+. The extent and rate of aggregation were highly dependent on Ca2+ concentration. Although both the rate and extent of aggregation at pH 7.5 were much lower than those at pH 5.5, aggregation of chromogranin A proceeded at both pH's. In this respect, chromogranin A differs from human chromogranin C which was shown by Gerdes et al. (Gerdes, H.-H., Rosa, P., Phillips, E., Baeuerle, P. A., Frank, R., Argos, P., and Huttner, W. B. (1989) J. Biol. Chem. 264, 12009-12015) to aggregate at pH 5.2 but not at pH 7.4.     

Inhibition by A23187 of conformational changes involved in the Ca(2+)-induced activation of sarcoplasmic reticulum Ca(2+)-ATPase.

We previously showed that A23187 in high ionophore/protein ratios almost completely inhibits the sarcoplasmic reticulum Ca(2+)-ATPase [Hara, H. & Kanazawa, T. (1986) J. Biol. Chem. 261, 16584-16590]. In an attempt to obtain information on the mechanism of this inhibition, the effects of A23187 on conformational changes involved in the Ca(2+)-induced activation of the enzyme were investigated. The purified enzyme from sarcoplasmic reticulum of rabbit skeletal muscle as well as the purified enzyme labeled with fluorescein 5-isothiocyanate (FITC) were preincubated with A23187 in the absence of Ca2+ at pH 7.0 and 0 degrees C for 45 min. The activation of the enzyme following addition of CaCl2 was assessed by determining the capacity for rapid formation of phosphoenzyme from ATP. This activation was strongly inhibited by the preincubation with A23187. This indicates that the previously observed inhibition of the Ca(2+)-ATPase is mostly due to hindrance of the Ca(2+)-induced activation of the enzyme. In the control, in which the FITC-labeled enzyme was preincubated without A23187, the fluorescence intensity of the bound FITC decreased in a biphasic manner upon addition of CaCl2. The first rapid phase of this fluorescence drop was unaffected by A23187, whereas its second slow phase was almost completely inhibited by this drug. These results show that the Ca(2+)-dependent conformational change is biphasic and that the second slow phase (but not the first rapid phase) of this conformational change is inhibited by A23187. This suggests that the observed inhibition of Ca2+ activation is attributed to hindrance of the second slow phase of the Ca(2+)-dependent conformational change.     

Structural basis for Ca(2+)-induced activation of human PAD4

Peptidylarginine deiminase 4 (PAD4) is a Ca(2+)-dependent enzyme that catalyzes the conversion of protein arginine residues to citrulline. Its gene is a susceptibility locus for rheumatoid arthritis. Here we present the crystal structure of Ca(2+)-free wild-type PAD4, which shows that the polypeptide chain adopts an elongated fold in which the N-terminal domain forms two immunoglobulin-like subdomains, and the C-terminal domain forms an alpha/beta propeller structure. Five Ca(2+)-binding sites, none of which adopt an EF-hand motif, were identified in the structure of a Ca(2+)-bound inactive mutant with and without bound substrate. These structural data indicate that Ca(2+) binding induces conformational changes that generate the active site cleft. Our findings identify a novel mechanism for enzyme activation by Ca(2+) ions, and are important for understanding the mechanism of protein citrullination and for developing PAD-inhibiting drugs for the treatment of rheumatoid arthritis.     

Human peroxisomal multifunctional enzyme type 2. Site-directed mutagenesis studies show the importance of two protic residues for 2-enoyl-CoA hydratase 2 activity

Beta-oxidation of acyl-CoAs in mammalian peroxisomes can occur via either multifunctional enzyme type 1 (MFE-1) or type 2 (MFE-2), both of which catalyze the hydration of trans-2-enoyl-CoA and the dehydrogenation of 3-hydroxyacyl-CoA, but with opposite chiral specificity. Amino acid sequence alignment of the 2-enoyl-CoA hydratase 2 domain in human MFE-2 with other MFE-2s reveals conserved protic residues: Tyr-347, Glu-366, Asp-370, His-406, Glu-408, Tyr-410, Asp-490, Tyr-505, Asp-510, His-515, Asp-517, and His-532. To investigate their potential roles in catalysis, each residue was replaced by alanine in site-directed mutagenesis, and the resulting constructs were tested for complementation in a yeast. After additional screening, the wild type and noncomplementing E366A and D510A variants were expressed and characterized. The purified proteins have similar secondary structural elements, with the same subunit composition. The E366A variant had a k(cat)/K(m) value 100 times lower than that of the wild type MFE-2 at pH 5, whereas the D510A variant was inactive. Asp-510 was imbedded in a novel hydratase 2 motif found in the hydratase 2 proteins. The data show that the hydratase 2 reaction catalyzed by MFE-2 requires two protic residues, Glu-366 and Asp-510, suggesting that their catalytic role may be equivalent to that of the two catalytic residues of hydratase 1.     

Molecular dynamics simulations revealed Ca(2+)-dependent conformational change of Calmodulin

Molecular dynamics simulations were performed to simulate Ca(2+)-dependent conformational change of calmodulin (CaM). Simulations of the fully Ca(2+)-bound form of CaM (Holo-CaM) and the Ca(2+)-free form (Apo-CaM) were performed in solution for 4 ns starting from the X-ray crystal structure of Holo-CaM. A striking difference was observed between the trajectories of Holo-CaM and Apo-CaM: the central helix remained straight in the former but became largely bent in the latter. Also, the flexibility of Apo-CaM was higher than that of Holo-CaM. The results indicated that the bound Ca(2+) ions harden the structure of CaM.     

Cytosolic Ca(2+) controls the loading dependence of IP(3)-induced Ca(2+) release.

It is still debated whether inositol 1,4, 5-trisphosphate(IP(3))-induced Ca(2+) release is loading-dependent. We now report that stimulation of the IP(3) receptor by luminal Ca(2+) depends on the cytosolic [Ca(2+)] in permeabilized A7r5 cells. The EC(50) and maximal extent of Ca(2+) release were loading-dependent in the presence of 5 mM 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid: the EC(50) increased 1.9-fold and the maximal release decreased from 88 to 52% when the stores contained 73% less Ca(2+). In the presence of 0.3 microM free Ca(2+), the EC(50) for filled and less filled stores differed, however, only 1.2-fold and the maximal Ca(2+) release was respectively 96 and 87% of the total releasable Ca(2+). At 1 microM free Ca(2+), the difference in EC(50) between filled and less filled stores again became larger (2.2-fold) and the maximal Ca(2+) release decreased from 93 to 87% when the stores contained less Ca(2+).     

Site-directed mutagenesis of human membrane-associated ganglioside sialidase: identification of amino-acid residues contributing to substrate specificity.

Unlike microbial sialidases, mammalian sialidases possess strict substrate specificity, for example the human membrane-associated sialidase, which hydrolyzes only gangliosides. To cast light on the molecular basis of this narrow substrate preference, predicted active site amino-acid residues of the human membrane sialidase were altered by site-directed mutagenesis. When compared with the active site amino-acid residues proposed for Salmonella typhimurium sialidase, only five out of 13 residues were found to be different to the human enzyme, these being located upstream of the putative transmembrane region. Alteration of seven residues, including these five, was followed by transient expression of the mutant enzymes in COS-1 cells and characterization of their kinetic properties using various substrates. Substitution of glutamic acid (at position 51) by aspartic acid and of arginine (at position 114) by glutamine or alanine resulted in retention of good catalytic efficiency toward ganglioside substrates, whereas other substitutions caused a marked reduction. The mutant enzyme E51D exhibited an increase in hydrolytic activity towards GM2 as well as sialyllactose (which are poor substrates for the wild-type) with change to a lower Km and a higher Vmax. R114Q demonstrated a substrate specificity shift in the same direction as E51D, whereas R114A enhanced the preference for gangliosides GD3 and GD1a that are effectively hydrolyzed by the wild-type. The inhibition experiments using 2-deoxy-2,3-didehydro-N-acetylneuraminic acid were consistent with the results in the alteration of substrate specificity. The findings suggest that putative active-site residues of the human membrane sialidase contribute to its substrate specificity.     

Trypanosoma brucei plasma membrane-type Ca(2+)-ATPase 1 (TbPMC1) and 2 (TbPMC2) genes encode functional Ca(2+)-ATPases localized to the acidocalcisomes and plasma membrane, and essential for Ca(2+) homeostasis and growth

Trypanosoma brucei adaptation and survival in its host involve integrated regulation of Ca(2+) pumps (Ca(2+)-ATPases), which are essential in calcium ion homeostasis. Here we report the cloning and sequencing of two genes (TbPMC1 and TbPMC2) encoding plasma membrane-type Ca(2+)-ATPases (PMCAs) of T. brucei, an agent of African trypanosomiasis. Indirect immunofluorescence analysis using antibodies against the proteins and against epitope tags introduced into each protein showed that TbPMC1 co-localized with the vacuolar H(+)-pyrophosphatase to the acidocalcisomes while TbPMC2 localized to the plasma membrane. Northern and Western blot analyses revealed that TbPMC1 and TbPMC2 are up-regulated during blood stages. TbPMC1 and TbPMC2 suppressed the Ca(2+) hypersensitivity of a mutant of S. cerevisiae that has a defect in vacuolar Ca(2+) accumulation. T. brucei Ca(2+)-ATPase genes were functionally characterized by using double-stranded RNA interference (RNAi) methodology to produce inducible Ca(2+)-ATPase-deficient procyclic forms. Similar results were obtained with bloodstream form trypomastigotes, except that the RNAi system was leaky and mRNA and protein levels recovered with time. The induction of dsRNA (RNAi) caused gross morphological alterations, and growth inhibition of procyclic forms. Induction of RNAi against TbPMC1 but not against TbPMC2 caused elevated levels of cytosolic Ca(2+) and decreased mobilization of Ca(2+) from intracellular stores following ionophore addition. These results establish that T. brucei PMCA-Ca(2+)-ATPases are essential for parasite viability and validate them as targets for drug development.     

Site-directed mutagenesis of beta-galactosidase (E. coli) reveals that tyr-503 is essential for activity

By using the technique of site-directed mutagenesis we have succeeded in replacing tyr-503 of beta-galactosidase (E. coli) with a phe. A study of the kinetic and stability properties of this mutant enzyme (F-503 beta-galactosidase) showed that the loss in activity upon this change is due to the loss of a catalytic group (rather than a detrimental change in the enzyme's overall structure or a change in the enzyme's binding capacity). This confirms previous suggestions that this tyr residue is involved in catalysis.     

Ca(2+) dynamics in the lumen of the endoplasmic reticulum in sensory neurons: direct visualization of Ca(2+)-induced Ca(2+) release triggered by physiological Ca(2+) entry

In cultured rat dorsal root ganglia neurons, we measured membrane currents, using the patch-clamp whole-cell technique, and the concentrations of free Ca(2+) in the cytosol ([Ca(2+)](i)) and in the lumen of the endoplasmic reticulum (ER) ([Ca(2+)](L)), using high- (Fluo-3) and low- (Mag-Fura-2) affinity Ca(2+)-sensitive fluorescent probes and video imaging. Resting [Ca(2+)](L) concentration varied between 60 and 270 microM. Activation of ryanodine receptors by caffeine triggered a rapid fall in [Ca(2+)](L) levels, which amounted to only 40--50% of the resting [Ca(2+)](L) value. Using electrophysiological depolarization, we directly demonstrate the process of Ca(2+)-induced Ca(2+) release triggered by Ca(2+) entry through voltage-gated Ca(2+) channels. The amplitude of Ca(2+) release from the ER lumen was linearly dependent on I(Ca).     

InsP(3)-induced Ca(2+) release in permeabilized invertebrate photoreceptors: a link between phototransduction and Ca(2+) stores

Using the low-affinity fluorescent Ca(2+) indicators, Mag-Fura-2 and Mag-Fura Red, we studied light- and InsP(3)-induced Ca(2+) release in permeabilized microvillar photoreceptors of the medicinal leech, Hirudo medicinalis. Two major components of the phosphoinositide signaling pathway, phospholipase-C and the InsP(3) receptor, were characterized immunologically and appropriately localized in photoreceptors. Whereas phospholipase-C was abudantly expressed in photoreceptive microvilli, InsP(3) receptors were found mostly in submicrovillar endoplasmic reticulum (SER). Permeabilization of the peripheral plasma membrane with saponin allowed direct measurements of luminal free Ca(2+) concentration (Ca(L)) changes. Confocal Ca(2+) imaging using Mag-Fura Red demonstrated that Ins(1,4,5)P(3) mobilizes Ca(2+) from SER. As detected with Mag-Fura-2, a brief 50ms light flash activated rapid Ca(2+) depletion of SER, followed by an effective refilling within 1min of dark adaptation after the light flash. Sensitivity to Ins(1,4,5)P(3) of the Ca(2+) release from SER in leech photoreceptors was accompanied by irreversible uncoupling of phototransduction from Ca(2+) release. Depletion of Ca(2+) stores was induced by Ins(1,4,5)P(3)(EC(50)= 4.75 microM) and the hyper-potent agonist adenophostin A (EC(50)/40nM) while the stereoisomer L-myo Ins(1,4,5)P(3) was totally inactive. Ins(1,4,5)P(3)- or adenophostin A-induced Ca(2+) release was inhibited by 0.1-1mg/ml heparin. The Ca(2+) pump inhibitors, cyclopiazonic acid and thapsigargin, in the presence of Ins(1,4,5)P(3), completely depleted Ca(2+) stores in leech photoreceptors.     

Alanine-scanning mutagenesis of HM-1 killer toxin and the essential residues for killing activity

Each of the aromatic, acidic and basic amino acid residues in HM-1 were separately substituted with alanine by site-directed mutagenesis. The mutant genes were successfully expressed in HM-1 resistant Saccharomyces cerevisiae. HM-1 gene analogues corresponding to the aromatic substitutions resulted in lower production of HM-1 analogues. In the case of the acidic amino acid residue and basic amino acid residue substitutions, some analogues were produced in the same amount as and exhibited similar killing activity to that of the wild type HM-1. But the H35A HM-1 analogue had completely lost the killing activity, and D44A, K21A, K46A, R82A, R85A and R86A HM-1 showed highly decreased killing activities. These results strongly indicate the importance of histidine-35, aspartic acid-44, lysine-21, lysine-46, and C-terminal arginine residues in HM-1 for the killing activity.     

Site-directed mutagenesis of the yeast resolving enzyme Cce1 reveals catalytic residues and relationship with the intron-splicing factor Mrs1

The Holliday junction-resolving enzyme Cce1 is a magnesium-dependent endonuclease, responsible for the resolution of recombining mitochondrial DNA molecules in Saccharomyces cerevisiae. We have identified a homologue of Cce1 from Candida albicans and used a multiple sequence alignment to predict residues important for junction binding and catalysis. Twelve site-directed mutants have been constructed, expressed, purified, and characterized. Using this approach, we have identified basic residues with putative roles in both DNA recognition and catalysis of strand scission and acidic residues that have a purely catalytic role. We have shown directly by isothermal titration calorimetry that a group of acidic residues vital for catalytic activity in Cce1 act as ligands for the catalytic magnesium ions. Sequence similarities between the Cce1 proteins and the group I intron splicing factor Mrs1 suggest the latter may also possess a binding site for magnesium, with a putative role in stabilization of RNA tertiary structure or catalysis of the splicing reaction.     

Site-directed mutagenesis of human endothelial cell ecto-ADPase/soluble CD39: requirement of glutamate 174 and serine 218 for enzyme activity and inhibition of platelet recruitment

Endothelial cell CD39/ecto-ADPase plays a major role in vascular homeostasis. It rapidly metabolizes ADP released from stimulated platelets, thereby preventing further platelet activation and recruitment. We recently developed a recombinant, soluble form of human CD39, solCD39, with enzymatic and biological properties identical to CD39. To identify amino acids essential for enzymatic/biological activity, we performed site-directed mutagenesis within the four highly conserved apyrase regions of solCD39. Mutation of glutamate 174 to alanine (E174A) and serine 218 to alanine (S218A) resulted in complete and approximately 90% loss of solCD39 enzymatic activity, respectively. Furthermore, compared to wild-type, S57A exhibited a 2-fold increase in ADPase activity without change in ATPase activity, while the tyrosine 127 to alanine (Y127A) mutant lost 50-60% of both ADPase and ATPase activity. The ADPase activity of wild-type solCD39 and each mutant, except for R135A, was greater with calcium as the required divalent cation than with magnesium, but for ATPase activity generally no such preference was observed. Y127A demonstrated the highest calcium/magnesium ADPase activity ratio, 2.8-fold higher than that of wild-type, even though its enzyme activity was greatly reduced. SolCD39 mutants were further characterized by correlating enzymatic with biological activity in an in vitro platelet aggregation system. Each solCD39 mutant was similar to wild-type in reversing platelet aggregation, except for E174A and S218A. E174A, completely devoid of enzymatic activity, failed to inhibit platelet responsiveness, as anticipated. S218A, with 91% loss of ADPase activity, could still reverse platelet aggregation, albeit much less effectively than wild-type solCD39. Thus, glutamate 174 and serine 218 are essential for both the enzymatic and biological activity of solCD39.