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At least 19 recordsLinked to original sources
PubMed2002

Functional analysis of calcium-binding EF-hand motifs of visinin-like protein-1.

Visinin-like protein-1 (VILIP-1), a myristoylated calcium sensor protein with three EF-hand motifs, modulates adenylyl cyclase activity. It translocates to membranes when a postulated "calcium-myristoyl switch" is triggered by calcium-binding to expose its sequestered myristoyl moiety. We investigated the contributions of the EF-hand motifs to the translocation of VILIP-1 to membranes and to the modulation of adenylyl cyclase activity. Mutation of residues crucial for binding calcium within each one of the EF-hand motifs indicated that they all contributed to binding calcium. Simultaneous mutations of all of the three EF-hand motifs completely abolished VILIP-1's ability to bind calcium, attenuated but did not eliminate its modulation of adenylyl cyclase activity, and abolished its calcium-dependence for association with cellular membranes. These results show that the calcium-binding EF-hand motifs of VILIP-1 do not have an essential role in modulating adenylyl cyclase activity but instead have a structural role in activating the "calcium-myristoyl switch" of VILIP-1.

Adenylyl Cyclases↗
PubMed1997

Identification of the Ca(2+)-binding domains in reticulocalbin, an endoplasmic reticulum resident Ca(2+)-binding protein with multiple EF-hand motifs.

Reticulocalbin (RCN) is a member of the EF-hand Ca(2+)-binding protein family and is a luminal protein of the endoplasmic reticulum (ER) with a molecular weight of 44,000 [Ozawa, M. and Muramatsu, T. (1993) J. Biol. Chem. 268, 699-705]. Although RCN has six repeats of a domain containing an EF-hand motif, the varying degrees of divergence of the amino acid sequences of these domains from the EF-hand consensus sequences suggested that some domains might have lost their Ca(2+)-binding capability and adopted new functions. To identify the domains involved in Ca(2+)-binding, discrete domains of RCN were expressed in Escherichia coli, using the glutathione S-transferase fusion protein system. 45Ca2+ blot analysis of the resultant fusion proteins revealed that the first, fourth, fifth, and sixth domains bind Ca2+, however, the second and third ones do not. The fusion proteins containing all six domains, and the first and second domains, respectively, showed Ca(2+)-dependent increases in their electrophoretic mobilities, suggesting that Ca2+ induces a conformational change in reticulocalbin.

Amino Acid Sequence↗
PubMed2000

Critical determinants of Ca(2+)-dependent inactivation within an EF-hand motif of L-type Ca(2+) channels.

L-type (alpha(1C)) calcium channels inactivate rapidly in response to localized elevation of intracellular Ca(2+), providing negative Ca(2+) feedback in a diverse array of biological contexts. The dominant Ca(2+) sensor for such Ca(2+)-dependent inactivation has recently been identified as calmodulin, which appears to be constitutively tethered to the channel complex. This Ca(2+) sensor induces channel inactivation by Ca(2+)-dependent CaM binding to an IQ-like motif situated on the carboxyl tail of alpha(1C). Apart from the IQ region, another crucial site for Ca(2+) inactivation appears to be a consensus Ca(2+)-binding, EF-hand motif, located approximately 100 amino acids upstream on the carboxyl terminus. However, the importance of this EF-hand motif for channel inactivation has become controversial since the original report from our lab implicating a critical role for this domain. Here, we demonstrate not only that the consensus EF hand is essential for Ca(2+) inactivation, but that a four-amino acid cluster (VVTL) within the F helix of the EF-hand motif is itself essential for Ca(2+) inactivation. Mutating these amino acids to their counterparts in non-inactivating alpha(1E) calcium channels (MYEM) almost completely ablates Ca(2+) inactivation. In fact, only a single amino acid change of the second valine within this cluster to tyrosine (V1548Y) supports much of the functional knockout. However, mutations of presumed Ca(2+)-coordinating residues in the consensus EF hand reduce Ca(2+) inactivation by only approximately 2-fold, fitting poorly with the EF hand serving as a contributory inactivation Ca(2+) sensor, in which Ca(2+) binds according to a classic mechanism. We therefore suggest that while CaM serves as Ca(2+) sensor for inactivation, the EF-hand motif of alpha(1C) may support the transduction of Ca(2+)-CaM binding into channel inactivation. The proposed transduction role for the consensus EF hand is compatible with the detailed Ca(2+)-inactivation properties of wild-type and mutant V1548Y channels, as gauged by a novel inactivation model incorporating multivalent Ca(2+) binding of CaM.

Amino Acid Motifs↗
PubMed1993

Reticulocalbin, a novel endoplasmic reticulum resident Ca(2+)-binding protein with multiple EF-hand motifs and a carboxyl-terminal HDEL sequence.

A novel Ca(2+)-binding protein, tentatively designated reticulocalbin, has been identified and characterized. Reticulocalbin is a luminal protein of the endoplasmic reticulum with an M(r) of 44,000 as revealed by biochemical analysis and immunofluorescence staining. The cDNA of reticulocalbin encodes a protein of 325 amino acids with an amino-terminal signal sequence of 20 amino acids. The protein has six repeats of a domain containing the high affinity Ca(2+)-binding motif, the EF-hand. Although oxygen-containing amino acids important for the positioning of Ca2+ are conserved in all six domains, the conserved glycine residues in the central portion of the EF-hand motif are absent in three of them. Calcium blots showed that recombinant reticulocalbin expressed in bacterial cells binds Ca2+. The protein has the sequence His-Asp-Glu-Leu (HDEL) at its carboxyl terminus. This is similar to the Lys-Asp-Glu-Leu sequence, which serves as a signal to retain the resident proteins in the endoplasmic reticulum of animal cells. A mutant protein lacking the HDEL sequence produced by in vitro mutagenesis has been shown to be secreted into medium in transient expression assays.

Amino Acid Sequence↗
PubMed1995

Conformational changes and calcium binding by calretinin and its recombinant fragments containing different sets of EF hand motifs.

Four recombinant fragments, representing different sets of EF-hand motifs of rat calretinin (CR) (I-II, I-III, III-VI, IV-VI), were prepared, and their Ca2+ -induced conformational changes were compared with those of full-length recombinant CR. All fragments were able to bind calcium ions as shown by 45Ca2+ overlay method on nitrocellulose and fluorescence measurements. The intrinsic tryptophan fluorescence intensity (FI) of apo-CR reversibly increased about 3-fold upon addition of calcium, indicating a change of conformation. The FI of fragments I-II (Trp 25) and I-III (Trp 25 and 116) increased about 1.4-fold on calcium binding, but that of fragment III-VI (Trp 116) increased 3.5-fold. Calcium titration of CR monitored by Trp fluorescence intensity showed that recombinant CR and some fragments bound Ca2+ with high affinity (Kd below 0.4 microM) and with high cooperativity. An apparent Hill coefficient for Ca2+ -induced fluorescence changes in CR was about 3.7. CR bound to organomercurial-agarose Cys 101 and 266 did not form cystine. The fluorescence intensities of cysteine-linked fluorescent probes 5-iodoacetamidofluorescein and N-(1-pyreneiodoacetamide) were increased approximately 1.3-fold upon calcium binding by CR. These data indicate that CR binds Ca2+ with high affinity and cooperativity and that this binding induces a change of conformation that involves the interaction of different parts of the molecule. Taken together, our results suggest that CR works as an on/off switch within a narrow range of free Ca2+ by interacting with as yet unidentified targets.

Animals↗
PubMed2001

Variability of calcium binding to EF-hand motifs probed by electrospray ionization mass spectrometry.

The modulation of calcium binding by the EF-hand motifs present in a calmodulin (CAM) homologue, a calcium binding protein (CaBP) from Entamoeba histolytica by three external parameters-pH, ligand coordinator EGTA, and fragmentor voltage was investigated by mass spectrometry. Calcium binding follows expected patterns at highly acidic and alkaline pH with the preponderance of the apo and the completely saturated forms, respectively. Surprisingly, additional nonspecific binding is observed near neutral pH. Studies on EGTA chelation and effects of fragmentor voltage showed cooperativity in calcium removal in at least one of the domains. Similar studies on a smaller construct containing the two high affinity carboxy terminal sites revealed interesting differences and provided an estimate of the specificity and tolerance of the EF-hand motifs to calcium binding and removal.

Calcium↗
PubMed1995

Structure of the gene encoding mouse reticulocalbin, a novel endoplasmic reticulum-resident Ca(2+)-binding protein with multiple EF-hand motifs.

Reticulocalbin, an endoplasmic reticulum (ER)-resident Ca(2+)-binding protein, is a member of the EF-hand Ca(2+)-binding protein superfamily [Ozawa, M. and Muramatsu, T. (1993) J. Biol. Chem. 268, 699-705]. Reticulocalbin has six repeats of a domain containing the EF-hand motif. In addition, the protein has an amino-terminal leader sequence which serves as a signal for transfer of the protein into the lumen of the ER, and a His-Asp-Glu-Leu sequence at its carboxy terminus which functions as a signal to retain the protein in the ER. In this paper, we describe the genomic structure of this unique Ca(2+)-binding protein. Southern blot analysis of mouse genomic DNA revealed that there is a single copy of the reticulocalbin gene per haploid genome. The gene spans over 13 kilobase pairs and encodes six separate exons. Thus, reticulocalbin differs from the cytosolic Ca(2+)-binding protein calbindin D28 which also has six EF-hand motif domains, but the gene for which is divided into 11 exons. While there is some correlation between exon division and protein domain structure, these relationships are not as clear as they are in other genes. Comparison of the gene organization of reticulocalbin with that of other EF-hand proteins revealed that reticulocalbin diverged very early from other members of the EF-hand protein super-family.

Amino Acid Sequence↗
PubMed1997

EF-hand motifs of alpha, beta and gamma isoforms of diacylglycerol kinase bind calcium with different affinities and conformational changes.

The three diacylglycerol kinase isoenzymes (DGK alpha, DGK beta and DGK gamma) cloned so far contain in common a tandem repeat of EF-hand motifs. However, the Ca2+ dependences of the DGK activities are known to be variable between isoenzymes, and the Ca(2+)-binding activities of these motifs have not been tested except for those present in DGK alpha. We therefore attempted to define the intrinsic properties of EF-hands occurring in the DGK isoenzymes. For this purpose we bacterially expressed and purified the EF-hand motifs (termed DKE forms) of the three DGKs. Equilibrium dialysis with the purified DKE forms showed that all of the expressed proteins could bind approx. 2 mol of Ca2+ per mol. However, the apparent dissociation constant (Kd) for calcium binding to alpha-DKE (9.9 microM) was an order of magnitude greater than those estimated for beta-DKE (0.89 microM) and gamma-DKE (0.40 microM). Experiments with 2-p-toluidinyl-naphthalene 6-sulphonate, a probe for hydrophobic regions of proteins, showed that the binding of Ca2+ to beta-DKE resulted in the exposure of hydrophobic amino acids, whereas hydrophobic regions of alpha-DKE and gamma-DKE were masked by the addition of Ca2+. Taken together, these results indicate that DGK alpha, DGK beta and DGK gamma possess EF-hand structures with intrinsic properties different from each other with respect to affinities for Ca2+ and Ca(2+)-induced conformational changes.

Amino Acid Sequence↗
PubMedDate not supplied

Molecular cloning of rat Spergen-3, a spermatogenic cell-specific gene-3, encoding a novel 75-kDa protein bearing EF-hand motifs.

By use of differential display in combination with cDNA cloning approach, we isolated a novel rat gene designated as Spergen-3, which has an open reading frame of 2055-length nucleotides encoding a protein of 685 amino acids. Spergen-3 gene is composed of 15 exons and mapped on chromosome 5q36, and its mouse counterpart, which shares 85.5% identity to rat Spergen-3 at the amino acid level, is mapped on chromosome 4E1. Spergen-3 encodes a 75-kDa soluble protein bearing putative 2 EF-hand motifs, proline-repeat, and a putative nuclear localization signal. Of the 2 EF-hand motifs in Spergen-3, the second one seems to match the consensus sequence. Reverse transcription-polymerase chain reaction analysis showed that the expression of Spergen-3 is developmentally up-regulated and that it is exclusively expressed in testis. In situ hybridization revealed that Spergen-3 mRNA was exclusively expressed in haploid spermatids, but its signal was weak or undetectable in spermatogonia, spermatocytes, and Sertoli cells as well as in interstitial cells. We interpreted these data as a potential role of Spergen-3, a new member of EF-hand family, in differentiation of haploid spermatids in testis.

Amino Acid Sequence↗
PubMed2004

Engineering strontium binding affinity in an EF-hand motif: a quantum chemical and molecular dynamics study.

Proteins with the ability to specifically bind strontium would potentially be of great use in the field of nuclear waste management. Unfortunately, no such peptides or proteins are known -- indeed, it is uncertain whether they exist under natural conditions due to low environmental concentrations of strontium. To investigate the possibility of devising such molecules, one of us (CV), in a previous experimental study, proposed starting from an EF-hand motif of the protein calmodulin and mutating some residues to change the motif's specificity for calcium into one for strontium. In this paper, which represents a theoretical complement to the experimental work, we analyzed small-molecule crystallographic structures and performed quantum chemical calculations to identify possible mutations. We then constructed seven mutant sequences of the EF-hand motif and analyzed their dynamical and binding behaviors using molecular dynamics simulations and free-energy calculations (using the MM/PBSA method). As a result of these analyzes we were able to isolate some characteristics that could lead to mutant peptides with enhanced strontium affinity.

Amino Acid Motifs↗
PubMed2001

Contribution of potential EF hand motifs to the calcium-dependent gating of a mouse brain large conductance, calcium-sensitive K(+) channel.

1. The large conductance, calcium-sensitive K(+) channel (BK(Ca) channel) is a unique member of the K(+)-selective ion channel family in that activation is dependent upon both direct calcium binding and membrane depolarization. Calcium binding acts to dynamically shift voltage-dependent gating in a negative or left-ward direction, thereby adjusting channel opening to changes in cellular membrane potential. 2. We hypothesized that the intrinsic calcium-binding site within the BK(Ca) channel alpha subunit may contain an EF hand motif, the most common, naturally occurring calcium binding structure. Following identification of six potential sites, we introduced a single amino acid substitution (D/E to N/Q or A) at the equivalent of the -z position of a bona fide EF hand that would be predicted to lower calcium binding affinity at each of the six sites. 3. Using macroscopic current recordings of wild-type and mutant BK(Ca) channels in excised inside-out membrane patches from HEK 293 cells, we observed that a single point mutation in the C-terminus (Site 6, FLD(923)QD to N), adjacent to the 'calcium bowl' described by Salkoff and colleagues, shifted calcium-sensitive gating right-ward by 50--65 mV over the range of 2--12 microM free calcium, but had little effect on voltage-dependent gating in the absence of calcium. Combining this mutation at Site 6 with a similar mutation at Site 1 (PVD(81)EK to N) in the N-terminus produced a greater shift (70--90 mV) in calcium-sensitive gating over the same range of calcium. We calculated that these combined mutations decreased the apparent calcium binding affinity approximately 11-fold (129.5 microM vs. 11.3 microm) compared to the wild-type channel. 4. We further observed that a bacterially expressed protein encompassing Site 6 of the BK(Ca) channel C-terminus and bovine brain calmodulin were both able to directly bind (45)Ca(2+) following denaturation and polyacrylamide gel electrophoresis (e.g. SDS-PAGE). 5. Our results suggest that two regions within the mammalian BK(Ca) channel alpha subunit, with sequence similarities to an EF hand motif, functionally contribute to the calcium-sensitive gating of this channel.

Amino Acid Sequence↗
PubMed2003

KIC, a novel Ca2+ binding protein with one EF-hand motif, interacts with a microtubule motor protein and regulates trichome morphogenesis.

Kinesin-like calmodulin binding protein (KCBP) is a microtubule motor protein involved in the regulation of cell division and trichome morphogenesis. Genetic studies have shown that KCBP is likely to interact with several other proteins. To identify KCBP-interacting proteins, we used the C-terminal region of KCBP in a yeast two-hybrid screen. This screening resulted in the isolation of a novel KCBP-interacting Ca2+ binding protein (KIC). KIC, with its single EF-hand motif, bound Ca2+ at a physiological concentration. Coprecipitation with bacterially expressed protein and native KCBP, gel-mobility shift studies, and ATPase assays with the KCBP motor confirmed that KIC interacts with KCBP in a Ca2+-dependent manner. Interestingly, although both Ca2+-KIC and Ca2+-calmodulin were able to interact with KCBP and inhibit its microtubule binding activity, the concentration of Ca2+ required to inhibit the microtubule-stimulated ATPase activity of KCBP by KIC was threefold less than that required for calmodulin. Two KIC-related Ca2+ binding proteins and a centrin from Arabidopsis, which contain one and four EF-hand motifs, respectively, bound Ca2+ but did not affect microtubule binding and microtubule-stimulated ATPase activities of KCBP, indicating the specificity of Ca2+ sensors in regulating their targets. Overexpression of KIC in Arabidopsis resulted in trichomes with reduced branch number resembling the zwichel/kcbp phenotype. These results suggest that KIC modulates the activity of KCBP in response to changes in cytosolic Ca2+ and regulates trichome morphogenesis.

Adenosine Triphosphatases↗
PubMed1997

Characterization of a helix-loop-helix (EF hand) motif of silver hake parvalbumin isoform B.

Parvalbumins are a class of calcium-binding proteins characterized by the presence of several helix-loop-helix (EF-hand) motifs. It is suspected that these proteins evolved via intragene duplication from a single EF-hand. Silver hake parvalbumin (SHPV) consists of three EF-type helix-loop-helix regions, two of which have the ability to bind calcium. The three helix-loop-helix motifs are designated AB, CD, and EF, respectively. In this study, native silver hake parvalbumin isoform B (SHPV-B) has been sequenced by mass spectrometry. The sequence indicates that this parvalbumin is a beta-lineage parvalbumin. SHPV-B was cleaved into two major fragments, consisting of the ABCD and EF regions of the native protein. The 33-amino acid EF fragment (residues 76-108), containing one of the calcium ion binding sites in native SHPV-B, has been isolated and studied for its structural characteristics, ability to bind divalent and trivalent cations, and for its propensity to undergo metal ion-induced self-association. The presence of Ca2+ does not induce significant secondary structure in the EF fragment. However, NMR and CD results indicate significant secondary structure promotion in the EF fragment in the presence of the higher charge-density trivalent cations. Sedimentation equilibrium analysis results show that the EF fragment exists in a monomer-dimer equilibrium when complexed with La3+.

Amino Acid Sequence↗
PubMed1991

The regulatory role of EF-hand motifs of pig 80K diacylglycerol kinase as assessed using truncation and deletion mutants.

To elucidate the regulatory function of EF-hand motifs of pig 80K diacylglycerol (DG) kinase, we constructed and expressed several truncation and deletion mutants of the enzyme in E. coli or COS-7 cells. The bacterially expressed EF-hand region could bind Ca2+ and was suggested to undergo conformational change like calmodulin. A mutant enzyme lacking EF-hands lost Ca(2+)-binding activity, but could be fully activated by phosphatidylserine (PS) or deoxycholate in the absence of Ca2+. The full activation of the wild-type enzyme by PS, on the other hand, was totally dependent on Ca2+. Further, the wild-type enzyme expressed in COS-7 cells was exclusively soluble, whereas the EF-hand-deleted mutant was considerably associated with the membranes. The results suggest that under Ca(2+)-free condition, the EF-hand masks the PS-binding site of the DG kinase, and that the Ca(2+)-binding results in the exposure of the PS-binding site through the conformational change of the EF-hand region.

Animals↗
PubMed2001

A calcium-binding protein with four EF-hand motifs in Streptomyces ambofaciens.

A gene (cabA) encoding a calcium-binding protein was cloned from Streptomyces ambofaciens. CabA was 180 amino acid residues long and contained four typical EF-hand motifs bearing high sequence similarity to the calcium-binding sites in calmodulin. Consistent with this, CabA showed distinct calcium-binding activity, comparable to bovine brain calmodulin. cabA was transcribed throughout growth, as found by S1 nuclease mapping. Southern hybridization experiments showed that a single copy of cabA was present in various Streptomyces species. A hypothetical relationship between CabA and aerial mycelium formation in this strain was examined, since S. ambofaciens showed calcium-dependent aerial mycelium formation. However, disruption of cabA or overexpression of cabA in S. ambofaciens caused no detectable phenotypic changes.

Amino Acid Sequence↗
PubMed1999

Involvement of EF hand motifs in the Ca(2+)-dependent binding of the pleckstrin homology domain to phosphoinositides.

The pleckstrin homology (PH) domains of phospholipase C (PLC)-delta1 and a related catalytically inactive protein, p130, both bind inositol phosphates and inositol lipids. The binding to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] by PLC-delta1 is proposed to be the critical interaction required for membrane localization to where the substrate resides; it is also required for the Ca(2+)-dependent activation of PLC-delta1 observed in the permeabilized cells. In the proximity of the PH domain, both PLC-delta1 and p130 possess the EF-hand domain, containing classical motifs implicated in calcium binding. Therefore, in the present study we examined whether the binding of the PH domain to PtdIns(4,5)P2 is regulated by changes in free Ca2+ concentration within the physiological range. A Ca2+ dependent increase in the binding to PtdIns(4,5)P2 was observed with a full-length PLC-delta1, while the isolated PH domain did not show any Ca2+ dependence. However, the connection of the EF-hand motifs to the PH domain restored the Ca2+ dependent increase in binding, even in the absence of the C2 domain. The p130 protein showed similar properties to PLC-delta1, and the EF-hand motifs were again required for the PH domain to exhibit a Ca2+ dependent increase in the binding to PtdIns(4,5)P2. The isolated PH domains from several other proteins which have been demonstrated to bind PtdIns(4,5)P2 showed no Ca2+ dependent enhancement of binding. However, when present within a chimera also containing PLC-delta1 EF-hand motifs, the Ca2+ dependent binding was again observed. These results suggest that the binding of Ca2+ to the EF-hand motifs can modulate binding to PtdIns(4,5)P2 mediated by the PH domain.

Blood Proteins↗
PubMed2001

Identification and characterization of an Entamoeba histolytica upstream regulatory element 3 sequence-specific DNA-binding protein containing EF-hand motifs.

The hgl5 gene of Entamoeba histolytica is negatively regulated through the upstream regulatory element 3 (URE3) DNA motif TATTCTATT. This motif is also present and significant in the function of the E. histolytica fdx gene promoter. A yeast one-hybrid screen was used to identify an E. histolytica cDNA encoding a protein (URE3-BP) that recognized this DNA motif. Analysis of the predicted amino acid sequence demonstrated the presence of two EF-hand motifs but identified no canonical DNA binding motifs. URE3-BP, expressed in bacteria, demonstrated Ca(2+)-dependent and sequence-specific recognition of the URE3 DNA sequence as assessed by electrophoretic mobility shift assays. Antibodies raised against URE3-BP blocked the formation of the URE3 DNA-protein complex by native nuclear extracts. The URE3-BP protein was present in the E. histolytica nucleus and cytoplasm with an apparent molecular mass of 22.6 kDa. Our results represent the first use of a yeast genetic screen to identify, on the basis of function, a DNA-binding protein of an early branching eukaryote. Since the URE3 DNA can modulate gene expression in both a positive and negative manner, this protein may have more than one mechanism of interaction with transcriptional machinery. Characterization of URE3-BP should provide insight into transcription regulation and virulence control in this parasite.

Amino Acid Sequence↗
PubMed1999

Calcium binding to the class I alpha-1,2-mannosidase from Saccharomyces cerevisiae occurs outside the EF hand motif.

Class I alpha-1,2-mannosidases are a family of Ca2+-dependent enzymes that have been conserved through eukaryotic evolution. These enzymes contain a conserved putative EF hand Ca2+-binding motif and nine invariant acidic residues. The catalytic domain of the alpha-1, 2-mannosidase from Saccharomyces cerevisiae was expressed in Pichia pastoris and was shown by atomic absorption and equilibrium dialysis to bind one Ca2+ ion with high affinity (KD = 4 x 10(-)7 M). Ca2+ protected the enzyme from thermal denaturation. Mutation of the 1st and 12th residues of the putative EF hand Ca2+ binding loop (D121N, D121A, E132Q, E132V, and D121A/E132V) had no effect on Ca2+ binding, demonstrating that the EF hand motif is not the site of Ca2+ binding. In contrast, three invariant acidic residue mutants (D275N, E279Q, and E438Q) lost the ability to bind 45Ca2+ following nondenaturing polyacrylamide gel electrophoresis whereas D86N, E132Q, E503Q, and E526Q mutants exhibited binding of 45Ca2+ similar to the wild-type enzyme. The wild-type enzyme had a Km and kcat of 0.5 mM and 12 s-1, respectively. The Km of E526Q was greatly increased to 4 mM with a small reduction in kcat to 5 s-1 whereas the kcat values of D86N and E132Q(V) were greatly reduced (0.005-0.007 s-1) with a decrease in Km (0.07-0.3 mM). The E503Q mutant is completely inactive. Asp275, Glu279, and Glu438 are therefore required for Ca2+ binding whereas Asp86, Glu132, and Glu503 are required for catalysis.

Asparagine↗