The antithrombin P1 residue is important for target proteinase specificity but not for heparin activation of the serpin. Characterization of P1 antithrombin variants with altered proteinase specificity but normal heparin activation

Heparin has been proposed to conformationally activate the serpin, antithrombin, by making the reactive center loop P1 arginine residue accessible to proteinases. To evaluate this proposal, we determined the effect of mutating the P1 arginine on antithrombin's specificity for target and nontarget proteinases in both native and heparin-activated states of the serpin. As expected, mutation of the P1 arginine to tryptophan, histidine, leucine, and methionine converted the specificity of antithrombin from a trypsin inhibitor (k(assoc) = 2 x 10(5) M(-1) s(-1)) to a chymotrypsin inhibitor (k(assoc) = 10(3)-10(5) M(-1) s(-1)). However, heparin pentasaccharide activation increased the reactivity of the P1 variants with chymotrypsin or of the wild-type inhibitor with trypsin only 2-6-fold, implying that the P1 residue had similar accessibilities to these proteinases in native and activated states. Mutation of the P1 arginine greatly reduced k(assoc) for antithrombin inhibition of thrombin and factor Xa from 40- to 5000-fold, but heparin normally accelerated the reactions of the variant antithrombins with these enzymes to make them reasonably efficient inhibitors (k(assoc) = 10(3)-10(4) M(-1) s(-1)). Fluorescence difference spectra of wild-type and P1 tryptophan variant antithrombins showed that the P1 tryptophan exhibited fluorescence properties characteristic of a solvent-exposed residue which were insignificantly affected by heparin activation. Moreover, all P1 variant antithrombins bound heparin with approximately 2-3-fold higher affinities than the wild type. These findings are consistent with the P1 mutations disrupting a P1 arginine-serpin body interaction which stabilizes the native low-heparin affinity conformation, but suggest that this interaction is of low energy and unlikely to limit the accessibility of the P1 residue. Together, these findings suggest that the P1 arginine residue is similarly accessible to proteinases in both native and heparin-activated states of the serpin and contributes similarly to the specificity of antithrombin for thrombin and factor Xa in the two serpin conformational states. Consequently, determinants other than the P1 residue are responsible for enhancing the specificity of antithrombin for the two proteinases when activated by heparin.     

Role of P2 glycine in determining the specificity of antithrombin reaction with coagulation proteases

Structural data suggests that bulky hydrophobic residues at the S2-S4 sub-sites of factor Xa (fXa) restrict the preference of this pocket for small and non-polar residues like Gly at the P2 position of substrates and inhibitors. However, kinetic studies monitoring the cleavage specificity of 10-residue peptides by fXa have identified Phe as the most preferred P2 residue and Gln-Phe-Arg-Ser-Leu-Ser as the most preferred P3-P3′ residues for recognition by fXa. To determine whether this mechanism of specificity is also true for fXa reaction with antithrombin (AT), we prepared two AT mutants having either a Phe at the P2 or Gln-Phe-Arg-Ser-Leu-Ser at the P3-P3′ positions of the reactive center loop. Inhibition kinetic studies indicated that the reactivity of P2-Phe with fXa was significantly (∼5-fold) impaired, however, the P3-P3′ mutant exhibited 1.5-fold improved reactivity with the protease, suggesting cooperative effects between P3-P3′ residues influence the P2 specificity of AT. Substitution of Tyr-99 of fXa with a Gly dramatically impaired the reactivity of fXa with wild-type AT, but improved its reactivity with the serpin mutants in the absence, but not in the presence of pentasaccharide. AT with a P2-Phe inhibited thrombin with >150-fold impaired reactivity, however, the defect was restored by either pentasaccharide or by replacing Leu-99 of thrombin with a Gly. The P3-P3′ mutant rapidly inhibited factors VIIa and XIa independent of pentasaccharide. These results indicate that P2-Gly plays a key role in determining the S2 sub-site specificity and target protease selectivity of AT in circulation.     

A Pro to Gly mutation in the hinge of the arabinose-binding protein enhances binding and alters specificity. Sugar-binding and crystallographic studies

The L-arabinose-binding protein (ABP) of Escherichia coli consists structurally of two distinct globular domains connected by a hinge of three separate peptide segments. Arabinose is bound and completely sequestered within the deep cleft between the two domains. With reduced affinity, ABP also binds D-galactose (approximately 2-fold reduction) and D-fucose (approximately 40-fold reduction). Experiments have been conducted to explore the role in sugar binding of the hinge connecting the two domains of ABP. To increase the flexibility of the hinge region, a glycine was substituted for a proline at position 254 by site-directed mutagenesis. Unexpectedly, this mutation resulted in the dramatic enhancement of galactose binding over that of arabinose. The affinity of the mutant ABP for galactose increased by over 20-fold, while that for arabinose and fucose remained relatively unchanged. We have measured association and dissociation rates of the Gly-254 ABP with L-arabinose, D-galactose, and D-fucose and have determined the crystallographic structure of the protein complexed with each of the three sugars. Both the ligand-binding kinetic measurements and structure analysis indicate that the altered specificity is due to an effective increase in the rigidity of the hinge in the closed conformation which is induced upon galactose binding. Stabilizing contacts are formed between the strands of the hinge in the Gly-254 ABP when galactose is bound which are not found in complexes with the other sugars or the liganded wild-type protein.     

Diversity in proteinase specificity of thermophilic lactobacilli as revealed by hydrolysis of dairy and vegetable proteins

Ability of industrially relevant species of thermophilic lactobacilli strains to hydrolyze proteins from animal (caseins and β-lactoglobulin) and vegetable (soybean and wheat) sources, as well as influence of peptide content of growth medium on cell envelope-associated proteinase (CEP) activity, was evaluated. Lactobacillus delbrueckii subsp. lactis (CRL 581 and 654), L. delbrueckii subsp. bulgaricus (CRL 454 and 656), Lactobacillus acidophilus (CRL 636 and 1063), and Lactobacillus helveticus (CRL 1062 and 1177) were grown in a chemically defined medium supplemented or not with 1 % Casitone. All strains hydrolyzed mainly β-casein, while degradation of α_s-caseins was strain dependent. Contrariwise, κ-Casein was poorly degraded by the studied lactobacilli. β-Lactoglobulin was mainly hydrolyzed by CRL 656, CRL 636, and CRL 1062 strains. The L. delbrueckii subsp. lactis strains, L. delbrueckii subsp. bulgaricus CRL 656, and L. helveticus CRL 1177 degraded gliadins in high extent, while the L. acidophilus and L. helveticus strains highly hydrolyzed soy proteins. Proteinase production was inhibited by Casitone, the most affected being the L. delbrueckii subsp. lactis species. This study highlights the importance of proteolytic diversity of lactobacilli for rational strain selection when formulating hydrolyzed dairy or vegetable food products.     

Characterization of drug transport,ATP hydrolysis,and nucleotide trapping by the human ABCG2 multidrug transporter. Modulation of substrate specificity by a point mutation

The overexpression of the human ATP-binding cassette half-transporter, ABCG2 (placenta-specific ABC transporter, mitoxantrone resistance-associated protein, breast cancer resistance protein), causes multidrug resistance in tumor cells. An altered drug resistance profile and substrate recognition were suggested for wild-type ABCG2 and its mutant variants (R482G and R482T); the mutations were found in drug-selected tumor cells. In order to characterize the different human ABCG2 transporters without possible endogenous dimerization partners, we expressed these proteins and a catalytic center mutant (K86M) in Sf9 insect cells. Transport activity was followed in intact cells, whereas the ATP binding and hydrolytic properties of ABCG2 were studied in isolated cell membranes. We found that the K86M mutant had no transport or ATP hydrolytic activity, although its ATP binding was retained. The wild-type ABCG2 and its variants, R482G and R482T, showed characteristically different drug and dye transport activities; mitoxantrone and Hoechst 33342 were transported by all transporters, whereas rhodamine 123 was only pumped by the R482G and R482T mutants. In each case, ABCG2-dependent transport was blocked by the specific inhibitor, fumitremorgin C. A relatively high basal ABCG2-ATPase, inhibited by fumitremorgin C, was observed in all active proteins, but specific drug stimulation could only be observed in the case of R482G and R482T mutants. We found that ABCG2 is capable of a vanadate-dependent adenine nucleotide trapping. Nucleotide trapping was stimulated by the transported compounds in the R482G and R482T variants but not in the wild-type ABCG2. These experiments document the applicability of the Sf9 expression system for parallel, quantitative examination of the specific transport and ATP hydrolytic properties of different ABCG2 proteins and demonstrate significant differences in their substrate interactions.     

Role of ternary complexes, in which heparin binds both antithrombin and proteinase, in the acceleration of the reactions between antithrombin and thrombin or factor Xa

Oligosaccharides (10-20 monosaccharide units) with high affinity for antithrombin, as well as larger high-affinity heparin fractions (having relative molecular masses between 6,000 and 21,500), all markedly accelerated the inhibition of Factor Xa by antithrombin. Moreover, all high-affinity oligosaccharides and heparins enhanced, to a similar extent, the amount of free proteolytically modified antithrombin cleaved at the reactive bond by Factor Xa. In contrast, a minimum high-affinity heparin size of approximately 18 monosaccharide units was required to significantly accelerate the inactivation of thrombin by antithrombin and to enhance the production of modified antithrombin by this enzyme. All high-affinity fractions studied had similar affinities for antithrombin, as determined by fluorescence titrations. In competition experiments, binary complexes of antithrombin with octadecasaccharide or larger high-affinity heparins, but not with smaller oligosaccharides, displaced inactivated 125I-thrombin from matrix-linked low-affinity heparin. Moreover, similar binary complexes with 3H-labeled octadecasaccharide or larger chains, but not with smaller oligosaccharides, were capable of binding to matrix-linked inactivated thrombin. These results indicate that simultaneous binding of antithrombin and thrombin to high-affinity heparin is a prerequisite to the acceleration of the antithrombin-thrombin reaction and that the minimum heparin sequence capable of binding both proteins comprises approximately 18 monosaccharide units. Similar complex formation apparently is not required for the acceleration of the antithrombin-Factor Xa reaction.     

The N-terminal segment of antithrombin acts as a steric gate for the binding of heparin.

The binding of heparin causes a conformational change in antithrombin to give an increased heparin binding affinity and activate the inhibition of thrombin and factor Xa. The areas of antithrombin involved in binding heparin and stabilizing the interaction in the high-affinity form have been partially resolved through the study of both recombinant and natural variants. The role of a section of the N-terminal segment of antithrombin, residues 22-46 (segment 22-46), in heparin binding was investigated using rapid kinetic analysis of the protein cleaved at residues 29-30 by limited proteolysis with thermolysin. The cleaved antithrombin had 5.5-fold lowered affinity for heparin pentasaccharide and 1.8-fold for full-length, high-affinity heparin. It was shown that, although the initial binding of heparin is slightly enhanced by the cleavage, it dissociates much faster from the cleaved form, giving rise to the overall decrease in heparin affinity. This implies that the segment constituting residues 22-46 in the N terminus of antithrombin hinders access to the binding site for heparin, hence the increased initial binding for the cleaved form, whereas, when heparin is bound, segment 22-46 is involved in the stabilization of the binding interaction, as indicated by the increased dissociation constant. When the heparin pentasaccharide is bound to antithrombin prior to incubation with thermolysin, it protects the N-terminal cleavage site, implying that segment 22-46 moves to interact with heparin in the conformational change and thus stabilizes the complex.     

Critical role of the linker region between helix D and strand 2A in heparin activation of antithrombin

The binding of pentasaccharide heparin to antithrombin induces a conformational change that is transmitted to the reactive center loop and increases the rate of inhibition of factor Xa by approximately 300-fold. The mechanism of such transmission is not known. To test the role of residues 134-137, which link helix D to beta-sheet A, in this signal transduction, we created variant antithrombins in which we removed amino acids 134-137 stepwise and cumulatively. Although the deletions did not compromise the fundamental ability of antithrombin to bind to heparin or to inhibit target proteinases thrombin and factor Xa, they did largely decouple conformational changes in the heparin-binding site from conformational activation of the reactive center loop. Because the variant with only Ala(134) removed was as compromised as variants with larger deletions, yet the variant with Ser(137) removed was normal, we concluded that the length of the linker is less important than the precise interrelationship between residues in this region and other residues involved in conformational activation of antithrombin.     

Importance of the P4' residue in human granzyme B inhibitors and substrates revealed by scanning mutagenesis of the proteinase inhibitor 9 reactive center loop

The cytotoxic lymphocyte serine proteinase granzyme B induces apoptosis of abnormal cells by cleaving intracellular proteins at sites similar to those cleaved by caspases. Understanding the substrate specificity of granzyme B will help to identify natural targets and develop better inhibitors or substrates. Here we have used the interaction of human granzyme B with a cognate serpin, proteinase inhibitor 9 (PI-9), to examine its substrate sequence requirements. Cleavage and sequencing experiments demonstrated that Glu(340) is the P1 residue in the PI-9 RCL, consistent with the preference of granzyme B for acidic P1 residues. Ala-scanning mutagenesis demonstrated that the P4-P4' region of the PI-9 RCL is important for interaction with granzyme B, and that the P4' residue (Glu(344)) is required for efficient serpin-proteinase binding. Peptide substrates based on the P4-P4' PI-9 RCL sequence and containing either P1 Glu or P1 Asp were cleaved by granzyme B (k(cat)/K(m) 9.5 x 10(3) and 1.2 x 10(5) s(-1) M(-1), respectively) but were not recognized by caspases. A substrate containing P1 Asp but lacking P4' Glu was cleaved less efficiently (k(cat)/K(m) 5.3 x 10(4) s(-1) M(-1)). An idealized substrate comprising the previously described optimal P4-P1 sequence (Ile-Glu-Pro-Asp) fused to the PI-9 P1'-P4' sequence was efficiently cleaved by granzyme B (k(cat)/K(m) 7.5 x 10(5) s(-1) M(-1)) and was also recognized by caspases. This contrasts with the literature value for a tetrapeptide comprising the same P4-P1 sequence (k(cat)/K(m) 6.7 x 10(4) s(-1) M(-1)) and confirms that P' residues promote efficient interaction of granzyme B with substrates. Finally, molecular modeling predicted that PI-9 Glu(344) forms a salt bridge with Lys(27) of granzyme B, and we showed that a K27A mutant of granzyme B binds less efficiently to PI-9 and to substrates containing a P4' Glu. We conclude that granzyme B requires an extended substrate sequence for specific and efficient binding and propose that an acidic P4' substrate residue allows discrimination between early (high affinity) and late (lower affinity) targets during the induction of apoptosis.     

Probing the molecular basis of factor Xa specificity by mutagenesis of the serpin, antithrombin.

The molecular basis of the substrate and inhibitor specificity of factor Xa, the serine proteinase of the prothrombinase complex, was investigated by constructing two mutants of human antithrombin (HAT) in which the reactive site loop of the serpin from the P4-P4' site was replaced with the corresponding residues of the two factor Xa cleavage sites in prothrombin (HAT/Proth-1 and HAT/Proth-2). These mutants together with prethrombin-2, the smallest zymogen form of thrombin containing only the second factor Xa cleavage site, were expressed in mammalian cells, purified to homogeneity and characterized in kinetic reactions with factor Xa in both the absence and presence of cofactors; factor Va, high affinity heparin and pentasaccharide fragment of heparin. HAT/Proth-1 inactivated factor Xa approximately 3-4-fold better than HAT/Proth-2 in either the absence or presence of heparin cofactors. In the absence of a cofactor, factor Xa reacted with the HAT/Proth-2 and prethrombin-2 with similar second-order rate constants (approximately 2-3x10(2) M(-1)s(-1)). Pentasaccharide catalyzed the inactivation rate of factor Xa by the HAT mutants 300-500-fold. A similar 10(4)-10(5)-fold enhancement in the reactivity of factor Xa with prethrombin-2 and the HAT mutants was observed in the presence of the cofactors Va and heparin, respectively. Factor Va did not influence the reactivity of factor Xa with either one of the HAT mutants. These results suggest that (1) in the absence of a cofactor, the P4-P4' residues of HAT and prethrombin-2 primarily determine the specificity reactions with factor Xa, (2) factor Va binding to factor Xa is not associated with allosteric changes in the catalytic pocket of enzyme that would involve interactions with the P4-P4' binding sites, and (3) similar to allosteric activation of HAT by heparin, a role for factor Va in the prothrombinase complex may involve rearrangement of the residues surrounding the scissile bond of the substrate to facilitate its optimal docking into the catalytic pocket of factor Xa.     

Partial activation of antithrombin without heparin through deletion of a unique sequence on the reactive site loop of the serpin.

Native antithrombin (AT) has an inactive reactive site loop conformation unless it is activated by a unique pentasaccharide fragment of heparin (H(5)). Structural data suggests that this may be due to preinsertion of two N-terminal residues of the reactive site loop of the serpin into the A-beta-sheet of the molecule. Relative to alpha(1)-antitrypsin, the reactive site loop of AT has three additional residues, Arg(399), Val(400), and Thr(401), at the C-terminal P' end of the loop. To determine whether a longer reactive site loop of AT is responsible for loop preinsertion in the native conformation, mutants of the serpin were expressed in which these residues were individually or in combination deleted. Kinetic analysis suggested that deletion of two residues, Val(400) and Thr(401), changed the solution equilibrium of the serpin in favor of the active conformation, thereby enhancing the inhibition of factor Xa by an order of magnitude independent of H(5). Interestingly, the reactivity of this mutant with thrombin was impaired by the same order of magnitude in the absence, but not in the presence of H(5). These results suggest that a longer reactive site loop in AT is responsible for its inactive native conformation toward factor Xa, while at same time AT requires this feature to regulate the activity of thrombin.     

Examination, by 1H-n.m.r. spectroscopy, of the binding of a synthetic, high-affinity heparin pentasaccharide to human antithrombin III

Binding of a synthetic, high-affinity heparin pentasaccharide and of intact heparin to both native and elastase-modified human antithrombin III have been examined by 1H-n.m.r. spectroscopy. The pentasaccharide perturbs many protein resonances in the same way as does intact heparin. There are, however, differences that seem to arise both from fewer contacts in the heparin binding-site when the pentasaccharide binds and from dissimilar conformational changes in the protein. The resonance of the H-2 atom of the histidine, considered to be the N-terminal residue and to be located in the heparin binding-site, is strongly perturbed by heparin binding both to native and modified antithrombin. The pentasaccharide has little effect on this histidine in either protein. Resonances from two of the remaining four histidine units are sensitive to longer-range conformational changes, and show differences between binding of the two heparin species both in native and modified ATIII. It is concluded that the pentasaccharide only partly fills the heparin binding-site and does not produce a conformational change identical to that caused by intact heparin. This is particularly significant as regards the mechanism of action of heparin, because the synthetic pentasaccharide activates ATIII towards Factor Xa, but not towards thrombin.     

New insights on the specificity of heparin and heparan sulfate lyases from Flavobacterium heparinum revealed by the use of synthetic derivatives of K5 polysaccharide from E. coli. and 2-O-desulfated heparin

The capsular polysaccharide from E. Coli, strain K5 composed of ...-->4)beta-D-GlcA(1-->4)alpha-D-GlcNAc(1-->4)beta-D-GlcA (1-->..., chemically modified K5 polysaccharides, bearing sulfates at C-2 and C-6 of the hexosamine moiety and at the C-2 of the glucuronic acid residues as well as 2-O desulfated heparin were used as substrates to study the specificity of heparitinases I and II and heparinase from Flavobacterium heparinum. The natural K5 polysaccharide was susceptible only to heparitinase I forming deltaU-GlcNAc. N-deacetylated, N-sulfated K5 became susceptible to both heparitinases I and II producing deltaU-GlcNS. The K5 polysaccharides containing sulfate at the C-2 and C-6 positions of the hexosamine moiety and C-2 position of the glucuronic acid residues were susceptible only to heparitinase II producing deltaU-GlcNS,6S and deltaU,2S-GlcNS,6S respectively. These combined results led to the conclusion that the sulfate at C-6 position of the glucosamine is impeditive for the action of heparitinase I and that heparitinase II requires at least a C-2 or a C-6 sulfate in the glucosamine residues of the substrate for its activity. Iduronic acid-2-O-desulfated heparin was susceptible only to heparitinase II producing deltaU-GlcNS,6S. All the modified K5 polysaccharides as well as the desulfated heparin were not substrates for heparinase. This led to the conclusion that heparitinase II acts upon linkages containing non-sulfated iduronic acid residues and that heparinase requires C-2 sulfated iduronic acid residues for its activity.     

Evidence for a plasma inhibitor of the heparin accelerated inhibition of factor Xa by antithrombin III.

The ability of heparin fractions of different molecular weight to potentiate the action of antithrombin III against the coagulation factors thrombin and Xa has been examined in purified reaction mixtures and in plasma. Residual thrombin and Xa have been determined by their peptidase activities against the synthetic peptide substrates H-D-Phe-Pip-Arg-pNA and Bz-Ile-Gly-Arg-pNA. High molecular weight heparin fractions were found to have higher anticoagulant activities than low molecular weight heparin when studied with both thrombin and Xa incubation mixtures in purified mixtures and in plasma. The inhibition of thrombin by heparin fractions and antithrombin III was unaffected by other plasma components. However, normal human plasma contained a component that inhibited the heparin and antithrombin III inhibition of Xa particularly when the high molecular weight heparin fraction was used. Experiments using a purified preparation of platelet factor 4 suggested that the platelet-derived heparin-neutralizing protein was not responsible for the inhibition.     

Immunocytochemical localization of proteins P1, P2, P3 of glycine decarboxylase and of the selenoprotein PA of glycine reductase,all involved in anaerobic glycine metabolism of Eubacterium acidaminophilum

Antibodies raised against the glycine decarboxylase proteins P1, P2, P3, and the selenoprotein P_A, a component of the glycine reductase complex, were used for immunocytochemical localization experiments. Cells of Eubacterium acidaminophilum from logarithmic growth phase were fixed in the growth media with paraformaldehyde and glutaraldehyde. Fixed cells were embedded by the low-temperature procedure using Lowicryl K4M resin, and the protein A-gold technique was applied for the localization experiments. The vicinity of the cytoplasmic membrane contained about 27% of all gold particles when proteins P1 and P2 were to be localized, 50% for protein P_A, and 61% for protein P3. Double immunocytochemical labeling experiments gave evidence for the existence of a protein P1/P2 complex located predominantly in the cytoplasm, and a P3/P_A complex located at the cytoplasmic membrane. Only in very few instances the labels for proteins P3 and P1 were seen very close together in respective doublelabeling experiments. These results indicate that glycine decarboxylase does not occur in this organism as a complex consisting of all four proteins, but that protein P3, the atypical lipoamide dehydrogenase, takes part in both the glycine decarboxylase as well as in the glycine reductase reaction.     

Home ranges of ship rats in a small New Zealand forest as revealed by trapping and tracking

Abstract

The home ranges of 5 ship rats (Rattus r. rattus L.) in a small forest area near Palmerston North were determined for 7 months by concurrent cage-trapping and smoked paper tracking. Baited tracking platforms were over 20 times as effective as cage-trapping in obtaining location data, and home ranges revealed by tracking were on average 5 times the area of trap-revealed home ranges. All the rats were to some extent cage-trap shy. However, although cage-traps could not supply useful information on range boundaries or swift acknowledgment of boundary changes, centres of activity calculated from trap data were comparable with those from tracking data. Tracking rates of individuals were variable; it would be risky to assume that they accurately reflected intensity of use of any area over short periods or when rats may have been competing for baits. The rats had stable home ranges; 3 females had ranges predominantly exclusive to each other. The progressive removal of individuals from the study area effected the prompt expansion of adjacent ranges to include vacated areas; that is, range size was inversely related to rat density.     

Relevance of oestrone presentation to the specificity of the elicited antisera, as revealed by affinity separation

Polyclonal antisera raised against two different azobenzoyl-oestrone derivatives were analysed to investigate both the latency/intensity relationship of the immune response and the influence of antigen presentation on the specificity of the antisera elicited. Elongation of the azo-bridge of the hapten ([p(carboxyphenyl)-azo]-1,3,5[10]-oestratrien-3 ol-17 one) with a short aliphatic chain (4-amino-n-butyric acid) resulted in a marginal increase in the antibody yield, without affecting the time required to attain the maximum titre. The increased flexibility and mobility of the extended azo-bridge was shown to result in the appearance of antisera which cross-reacted with oestrogens with D ring structures different to that of oestrone. Antiserum fractionation by affinity chromatography through a stationary phase exposing the carrier protein determinants, as modified by the addition of the coupling bridge and the phenol ring, resulted in a reduction in its specificity. These findings are discussed with regard to the phenomena underlying specipecificity of a polyclonal antiserum.     

ATP-binding modes and functionally important interdomain bonds of sarcoplasmic reticulum Ca2+-ATPase revealed by mutation of glycine 438, glutamate 439, and arginine 678

ATP binds to sarcoplasmic reticulum Ca(2+)-ATPase both in a phosphorylating (catalytic) mode and in a nonphosphorylating (modulatory) mode, the latter leading to acceleration of phosphoenzyme turnover (Ca(2)E(1)P --> E(2)P and E(2)P --> E(2) reactions) and Ca(2+) binding (E(2) --> Ca(2)E(1)). In some of the Ca(2+)-ATPase crystal structures, Arg(678) and Glu(439) seem to be involved in the binding of nucleotide or an associated Mg(2+) ion. We have replaced Arg(678), Glu(439), and Gly(438) with alanine to examine their importance for the enzyme cycle and the modulatory effects of ATP and MgATP. The results point to the key role of Arg(678) in nucleotide binding and to the importance of interdomain bonds Glu(439)-Ser(186) and Arg(678)-Asp(203) in stabilizing the E(2)P and E(2) intermediates, respectively. Mutation of Arg(678) had conspicuous effects on ATP/MgATP binding to the E(1) form and ADP binding to Ca(2)E(1)P, as well as ATP/MgATP binding in modulatory modes to E(2)P and E(2), whereas the effects on ATP/MgATP acceleration of the Ca(2)E(1)P --> E(2)P transition were small, suggesting that the nucleotide that accelerates Ca(2)E(1)P --> E(2)P binds differently from that modulating the E(2)P --> E(2) and E(2) --> Ca(2)E(1) reactions. Mutation of Glu(439) hardly affected nucleotide binding to E(1), Ca(2)E(1)P, and E(2), but it led to disruption of the modulatory effect of ATP on E(2)P --> E(2) and acceleration of the latter reaction, indicating that ATP normally modulates E(2)P --> E(2) by interfering with the interaction between Glu(439) and Ser(186). Gly(438) seems to be important for this interaction as well as for nucleotide binding, probably because of its role in formation of the helix containing Glu(439) and Thr(441).