Probing the mechanism of inactivation of human pyruvate dehydrogenase by phosphorylation of three sites

Activity of the mammalian pyruvate dehydrogenase complex (PDC) is regulated by phosphorylation-dephosphorylation of three serine residues (designated site 1, Ser-264; site 2, Ser-271; site 3, Ser-203) in the alpha subunit of the pyruvate dehydrogenase (E1) component. Substitutions of the phosphorylation sites were generated by site-directed mutagenesis. Glutamate (S1E) and aspartate (S1D) substitutions at site 1 resulted in the complete loss of PDC activity; however, these mutants were variably active in the decarboxylation and 2,6-dichlorophenolindophenol assays. S1Q had only 3% of wild-type PDC activity. The apparent K(m) values for pyruvate increased for the mutants of site 1 when determined in the 2,6-dichlorophenolindophenol assay. The substitutions at sites 2 and 3 caused only moderate reductions in activity in the three assays. S3E had a 27-fold increase in the apparent K(m) for thiamine pyrophosphate and 8-fold increase in the K(i) for pyrophosphate. Site 3 was almost completely protected from phosphorylation by thiamine pyrophosphate. The results show that the size rather than negative charge of the substituted amino acid residue affects the active site of E1 and that modification of each of the three serine residues affect the active site in a site-specific manner for its ability to bind the cofactor and substrates.     

Involvement of lysine-193 of the conserved "K-T-G-G" motif in the catalysis of maize starch synthase IIa

It has been suggested that the lysine residue in the conserved K-T-G-G motif could be the substrate ADP-glucose binding site of Escherichia coli glycogen synthase (GS). Since the K-X-G-G motif is highly conserved between E. coli GS and all the maize starch synthase (SS) isozymes, it has become widely accepted that the lysine in the conserved K-T-G-G motif may also function as the ADPGlc binding site of maize SS. We have used chemical modification and site-directed mutagenesis to study the function of lysine residues in SS. Pyridoxal-5'-phosphate inactivated maize SSIIa activity in a time and concentration dependent manner. ADPGlc completely protected SSIIa from inactivation by pyridoxal-5'-phosphate, indicating that lysine residue(s) could be important for ADPGlc binding and enzyme catalysis. In contrast to E. coli GS, mutation of conserved lysine193 (K-T-G-G) in maize SS did not alter the ADPGlc binding while significantly changing the enzyme activity toward different primers. Our results suggest that lysine-193 (K-T-G-G) is not directly involved in ADPGlc binding, instead mutation in the conserved lysine position affected the primer preference.     

Site-directed mutagenesis of Escherichia coli acetylglutamate kinase and aspartokinase III probes the catalytic and substrate-binding mechanisms of these amino acid kinase family enzymes and allows three-dimensional modelling of aspartokinase

We test, using site-directed mutagenesis, predictions based on the X-ray structure of N-acetyl-L-glutamate kinase (NAGK), the paradigm of the amino acid kinase protein family, about the roles of specific residues on substrate binding and catalysis. The mutations K8R and D162E decreased V([sustrate]= infinity ) 100-fold and 1000-fold, respectively, in agreement with the predictions that K8 catalyzes phosphoryl transfer and D162 organizes the catalytic groups. R66K and N158Q increased selectively K(m)(Asp) three to four orders of magnitude, in agreement with the binding of R66 and N158 to the C(alpha) substituents of NAG. Mutagenesis in parallel of aspartokinase III (AKIII phosphorylates aspartate instead of acetylglutamate), another important amino acid kinase family member of unknown 3-D structure, identified in AKIII two residues, K8 and D202, that appear to play roles similar to those of K8 and D162 of NAGK, and supports the involvement of E119 and R198, similarly to R66 and N158 of NAGK, in the binding of the amino acid substrate, apparently interacting, respectively, with the alpha-NH(3)(+) and alpha-COO(-) of aspartate. These results and an improved alignment of the NAGK and AKIII sequences have guided us into 3-D modelling of the amino acid kinase domain of AKIII using NAGK as template. The model has good stereochemistry and validation parameters. It provides insight into substrate binding and catalysis, agreeing with mutagenesis results with another aspartokinase that were not considered when building the model.AKIII is homodimeric and is inhibited by lysine. Lysine may bind to a regulatory region that is C-terminal to the amino acid kinase domain. We make a C-terminally truncated AKIII (AKIIIt) and show that the C-region is involved in intersubunit interactions, since AKIIIt is found to be monomeric. Further, it is inactive, as demanded if dimer formation is essential for activity. Models for AKIII architecture are proposed that account for these findings.     

Recognition of the inner lipoyl-bearing domain of dihydrolipoyl transacetylase and of the blood glucose-lowering compound AZD7545 by pyruvate dehydrogenase kinase 2

Pyruvate dehydrogenase kinase 2 (PDHK2) is a unique mitochondrial protein kinase that regulates the activity of the pyruvate dehydrogenase multienzyme complex (PDC). PDHK2 is an integral component of PDC tightly bound to the inner lipoyl-bearing domains (L2) of the dihydrolipoyl transacetylase component (E2) of PDC. This association has been reported to bring about an up to 10-fold increase in kinase activity. Despite the central role played by E2 in the maintenance of PDHK2 functionality in the PDC-bound state, the molecular mechanisms responsible for the recognition of L2 by PDHK2 and for the E2-dependent PDHK2 activation are largely unknown. In this study, we used a combination of molecular modeling and site-directed mutagenesis to identify the amino acid residues essential for the interaction between PDHK2 and L2 and for the activation of PDHK2 by E2. On the basis of the results of site-directed mutagenesis, it appears that a number of PDHK2 residues located in its R domain (P22, L23, F28, F31, F44, L45, and L160) and in the so-called "cross arm" structure (K368, R372, and K391) are critical in determining the strength of the interaction between PDHK2 and L2. The residues of L2 essential for recognition by PDHK2 include L140, K173, I176, E179, and to a lesser extent D164, D172, and A174. Importantly, certain PDHK2 residues forming interfaces with L2, i.e., K17, P22, F31, F44, R372, and K391, are also critical for the maintenance of enhanced PDHK2 activity in the E2-bound state. Finally, evidence that the blood glucose-lowering compound AZD7545 disrupts the interactions between PDHK2 and L2 and thereby inhibits PDHK2 activity is presented.     

Role of the N-terminal starch-binding domains in the kinetic properties of starch synthase III from Arabidopsis thaliana

Starch synthase III (SSIII), one of the SS isoforms involved in plant starch synthesis, has been reported to play a regulatory role in the synthesis of transient starch. SSIII from Arabidopsis thaliana contains 1025 amino acid residues and has an N-terminal transit peptide for chloroplast localization which is followed by three repeated starch-binding domains (SBDs; SSIII residues 22-591) and a C-terminal catalytic domain (residues 592-1025) similar to bacterial glycogen synthase. In this work, we constructed recombinant full-length and truncated isoforms of SSIII, lacking one, two, or three SBDs, and recombinant proteins, containing three, two, or one SBD, to investigate the role of these domains in enzyme activity. Results revealed that SSIII uses preferentially ADPGlc, although UDPGlc can also be used as a sugar donor substrate. When ADPGlc was used, the presence of the SBDs confers particular properties to each isoform, increasing the apparent affinity and the V max for the oligosaccharide acceptor substrate. However, no substantial changes in the kinetic parameters for glycogen were observed when UDPGlc was the donor substrate. Under glycogen saturating conditions, the presence of SBDs increases progressively the apparent affinity and V max for ADPGlc but not for UDPGlc. Adsorption assays showed that the N-terminal region of SSIII, containing three, two, or one SBD module have increased capacity to bind starch depending on the number of SBD modules, with the D23 protein (containing the second and third SBD module) being the one that makes the greatest contribution to binding. The results presented here suggest that the N-terminal SBDs have a regulatory role, showing a starch binding capacity and modulating the catalytic properties of SSIII.     

A structure-function study of a proton transport pathway in the gamma-class carbonic anhydrase from Methanosarcina thermophila

Four glutamate residues in the prototypic gamma-class carbonic anhydrase from Methanosarcina thermophila (Cam) were characterized by site-directed mutagenesis and chemical rescue studies. Alanine substitution indicated that an external loop residue, Glu 84, and an internal active site residue, Glu 62, are both important for CO(2) hydration activity. Two other external loop residues, Glu 88 and Glu 89, are less important for enzyme function. The two E84D and -H variants exhibited significant activity relative to wild-type activity in pH 7.5 MOPS buffer, suggesting that the original glutamate residue could be substituted with other ionizable residues with similar pK(a) values. The E84A, -C, -K, -Q, -S, and -Y variants exhibited large decreases in k(cat) values in pH 7.5 MOPS buffer, but only exhibited small changes in k(cat)/K(m). These same six variants were all chemically rescued by pH 7.5 imidazole buffer, with 23-46-fold increases in the apparent k(cat). These results are consistent with Glu 84 functioning as a proton shuttle residue. The E62D variant exhibited a 3-fold decrease in k(cat) and a 2-fold decrease in k(cat)/K(m) relative to those of the wild type in pH 7.5 MOPS buffer, while other substitutions (E62A, -C, -H, -Q, -T, and -Y) resulted in much larger decreases in both k(cat) and k(cat)/K(m). Imidazole did not significantly increase the k(cat) values and slightly decreased the k(cat)/K(m) values of most of the Glu 62 variants. These results indicate a primary preference for a carboxylate group at position 62, and support a proposed catalytic role for residue Glu 62 in the CO(2) hydration step, but do not definitively establish its role in the proton transport step.     

Vesicular stomatitis virus glycoprotein mutations that affect membrane fusion activity and abolish virus infectivity.

We have introduced amino acid substitutions into two regions of the extracellular domain of the vesicular stomatitis virus (VSV) glycoprotein (G protein) and examined the effect of these mutations on protein transport, low-pH-induced stability of G protein oligomers, and membrane fusion activity. We suggested previously that the region between amino acids 118 and 139 may be important for the membrane fusion activity of G protein, on the basis of the characterization of a fusion-defective G protein mutant (M. A. Whitt, P. Zagouras, B. Crise, and J. K. Rose, J. Virol. 64:4907-4913, 1990). It has also been postulated by others that this region as well as the region between amino acids 181 and 212 may constitute putative internal fusion domains of VSV G protein. In this report, we show that three different amino acids substitutions between residues 118 and 139 (G-124-->E, P-127-->D, and A-133-->K) either altered or abolished low-pH-dependent membrane fusion activity. In contrast, substitutions between residues 192 and 212 resulted either in G proteins that had wild-type fusion activity or in mutant proteins in which the mutation prevented transport of G protein to the cell surface. Two of the substitutions between residues 118 and 139 (G-124-->E and P-127-->D) resulted in G proteins that were fusion defective at pH 5.7, although syncytia were observed after cells were treated with fusion buffer at pH 5.5, albeit at levels significantly less than that induced by wild-type G protein. Interestingly, when either G-124-->E or P-127-->D was incorporated into tsO45 virions, the resulting particles were not infectious, presumably because the viral envelope was not able to fuse with the proper intracellular membrane. These results support the hypothesis that the region between amino acids 118 and 139 is important for the membrane fusion activity of VSV G protein and may constitute an internal fusion domain.     

Enhanced transaminase activity of a bifunctional L-aspartate 4-decarboxylase

L-Aspartate 4-decarboxylase (Asd) catalyzes mainly the beta-decarboxylation of aspartate and also transamination with alpha-keto acids. To investigate residues that are critical in directing the reaction pathway, seven point mutations were designed based on the differences between Asd and amiontransferases in conservative amino acid residues. All mutant Asds were purified and characterized. The F204W mutant enhanced aminotransferase activity, and its ratio to beta-decarboxylase activity was 3.8-fold. Its K(m) values for aspartate and alpha-ketoglutarate were 1.3 and 0.17 mM, respectively, representing a large increase in the binding affinity with substrates. The K347R mutation did not increase transaminase activity. The D360P mutation decreased transaminase activity and was more specific in catalyzing beta-decarboxylation reaction. This is the first study that successfully increased transaminase activity in Asd via site-directed mutagenesis. The modeled protein structure reveals how the residue may involve in reaction specificity, providing insights into comprehending the molecular evolution of this bifunctional enzyme.     

Site-directed mutagenesis studies of rat steroid 5alpha-reductase (isozyme-1): mutation of residues in the cofactor binding and C-terminal regions

We have investigated the roles of highly conserved glycine (G175, G185), negatively charged (E188, D165) and histidine residues (H233, H237) in rat steroid 5alpha-reductase (isozyme-1), on NADPH, testosterone (T) binding and enzyme activity. The mutations G175R and G175S result in a two- to threefold increase in K(m)(NADPH) and an approximately fourfold decrease in the V(max) with no change in K(m)(T). The mutation G185W resulted in a fivefold decrease in K(m)(NADPH) and an eightfold decrease in V(max), with no change in K(m)(T), whereas the mutations E188Q and D165N both resulted in inactive enzyme. Steady-state kinetic measurements showed that the mutation H233R resulted in an approximately 40-fold decrease in V(max), an approximately 20-fold increase in K(m)(T) and no alteration in K(m)(NADPH), whereas the mutation H237R resulted in virtually inactive enzyme. The results suggest that the conserved glycines are not essential for cofactor binding and activity, and that the negatively charged residues may contribute to enzyme stability, whereas the C-terminal histidines appear to be involved in substrate binding and catalytic activity.     

Directed evolution relieves product inhibition and confers in vivo function to a rationally designed tyrosine aminotransferase

The Escherichia coli aspartate (AATase) and tyrosine (TATase) aminotransferases share 43% sequence identity and 72% similarity, but AATase has only 0.08% and 0.01% of the TATase activities (k(cat)/K(m)) for tyrosine and phenylalanine, respectively. Approximately 5% of TATase activity was introduced into the AATase framework earlier both by rational design (six mutations, termed HEX) and by directed evolution (9-17 mutations). The enzymes realized from the latter procedure complement tyrosine auxotrophy in TATase deficient E. coli. HEX complements even more poorly than does wild-type AATase, even though the (k(cat)/K(m)) value for tyrosine exhibited by HEX is similar to those of the enzymes found from directed evolution. HEX, however, is characterized by very low values of K(m) and K(D) for dicarboxylic ligands, and by a particularly slow release for oxaloacetate, the product of the reaction with aspartate and a TCA cycle intermediate. These observations suggest that HEX exists largely as an enzyme-product complex in vivo. HEX was therefore subjected to a single round of directed evolution with selection for complementation of tyrosine auxotrophy. A variant with a single amino acid substitution, A293D, exhibited substantially improved TATase function in vivo. The A293D mutation alleviates the tight binding to dicarboxylic ligands as K(m)s for aspartate and alpha-ketoglutarate are >20-fold higher in the HEX + A293D construct compared to HEX. This mutation also increased k(cat)/K(m)(Tyr) threefold. A second mutation, I73V, elicited smaller but similar effects. Both residues are in close proximity to Arg292 and the mutations may function to modulate the arginine switch mechanism responsible for dual substrate recognition in TATases and HEX.     

Carboxyl residues in the active site of human phenol sulfotransferase (SULT1A1)

The carboxyl-specific amino acid modification reagent, Woodward's reagent K (WK), was utilized to characterize carboxyl residues (Asp and Glu) in the active site of human phenol sulfotransferase (SULT1A1). SULT1A1 was purified using the pMAL-c2 expression system in E. coli. WK inactivated SULT1A1 activity in a time- and concentration-dependent manner. The inactivation followed first-order kinetics relative to both SULT1A1 and WK. Both phenolic substrates and adenosine 3'-phosphate 5'-phosphosulfate (PAPS) protected against the inactivation, which suggests the carboxyl residue modification causing the inactivation took place within the active site of the enzyme. With partially inactivated SULT1A1, both V(max) and K(m) changed for PAPS, while for phenolic substrates, V(max) decreased and K(m) did not change significantly. A computer model of the three-dimensional structure of SULT1A1 was constructed based on the mouse estrogen sulfotransferase (mSULT1E1) X-ray crystal structure. According to the model, Glu83, Asp134, Glu246, and Asp263 are the residues likely responsible for the inactivation of SULT1A1 by WK. According to these results, five SULT1A1 mutants, E83A, D134A, E246A, D263A, and E151A, were generated (E151A as control mutant). Specific activity determination of the mutants demonstrated that E83A and D134A lost almost 100% of the catalytic activity. E246A and D263A also decreased SULT1A1 activity, while E151A did not change SULT1A1 catalytic activity significantly. This work demonstrates that carboxyl residues are present in the active site and are important for SULT1A1 catalytic activity. Glu83 and E134 are essential amino acids for SULT1A1 catalytic activity.     

Substrate and inhibitor specificity of Mycobacterium avium dihydrofolate reductase

Dihydrofolate reductase (EC 1.5.1.3) is a key enzyme in the folate biosynthetic pathway. Information regarding key residues in the dihydrofolate-binding site of Mycobacterium avium dihydrofolate reductase is lacking. On the basis of previous information, Asp31 and Leu32 were selected as residues that are potentially important in interactions with dihydrofolate and antifolates (e.g. trimethoprim), respectively. Asp31 and Leu32 were modified by site-directed mutagenesis, giving the mutants D31A, D31E, D31Q, D31N and D31L, and L32A, L32F and L32D. Mutated proteins were expressed in Escherichia coli BL21(DE3)pLysS and purified using His-Bind resin; functionality was assessed in comparison with the recombinant wild type by a standard enzyme assay, and growth complementation and kinetic parameters were evaluated. All Asp31 substitutions affected enzyme function; D31E, D31Q and D31N reduced activity by 80-90%, and D31A and D31L by > 90%. All D31 mutants had modified kinetics, ranging from three-fold (D31N) to 283-fold (D31L) increases in K(m) for dihydrofolate, and 12-fold (D31N) to 223 077-fold (D31L) decreases in k(cat)/K(m). Of the Leu32 substitutions, only L32D caused reduced enzyme activity (67%) and kinetic differences from the wild type (seven-fold increase in K(m); 21-fold decrease in k(cat)/K(m)). Only minor variations in the K(m) for NADPH were observed for all substitutions. Whereas the L32F mutant retained similar trimethoprim affinity as the wild type, the L32A mutation resulted in a 12-fold decrease in affinity and the L32D mutation resulted in a seven-fold increase in affinity for trimethoprim. These findings support the hypotheses that Asp31 plays a functional role in binding of the substrate and Leu32 plays a functional role in binding of trimethoprim.     

Identification of amino acid residues responsible for taurocyamine binding in mitochondrial taurocyamine kinase from Arenicola brasiliensis

In order to investigate the residues associated with binding of the substrate taurocyamine in Arenicola mitochondrial taurocyamine kinase (TK), we performed Ala-scanning of the amino acid sequence HTKTV at positions 67-71 on the GS loop, and determined apparent K(m) and V(max) (appK(m) and appV(max), respectively) of the mutant forms for the substrates taurocyamine and glycocyamine. The appK(m) values for taurocyamine of the K69A, T70A and V71A mutants were significantly increased as compared with wild-type, suggesting that these residues are associated with taurocyamine binding. Of special interest is a property of V71A mutant: its catalytic efficiency for glycocyamine was twice that for taurocyamine, indicating that the V71A mutant acts like a glycocyamine kinase, rather than a TK. The role of the amino acid residue K95 of Arenicola MiTK was also examined. K95 was replaced with R, H, Y, I, A and E. K95R, K95H and K95I have a 3-fold higher affinity for taurocyamine, and activity was largely lost in K95E. On the other hand, the K95Y mutant showed a rather unique feature; namely, an increase in substrate concentration caused a decrease in initial velocity of the reaction (substrate inhibition). This is the first report on the key amino acid residues responsible for taurocyamine binding in mitochondrial TK.     

Human ferrochelatase: characterization of substrate-iron binding and proton-abstracting residues

The terminal step in heme biosynthesis, the insertion of ferrous iron into protoporphyrin IX to form protoheme, is catalyzed by the enzyme ferrochelatase (EC 4.99.1.1). A number of highly conserved residues identified from the crystal structure of human ferrochelatase as being in the active site were examined by site-directed mutagenesis. The mutants Y123F, Y165F, Y191H, and R164L each had an increased K(m) for iron without an altered K(m) for porphyrin. The double mutant R164L/Y165F had a 6-fold increased K(m) for iron and a 10-fold decreased V(max). The double mutant Y123F/Y191F had low activity with an elevated K(m) for iron, and Y123F/Y165F had no measurable activity. The mutants H263A/C/N, D340N, E343Q, E343H, and E343K had no measurable enzyme activity, while E343D, E347Q, and H341C had decreased V(max)s without significant alteration of the K(m)s for either substrate. D340E had near-normal kinetic parameters, while D383A and H231A had increased K(m)s for iron. On the basis of these data and the crystal structure of human ferrochelatase, it is proposed that residues E343, H341, and D340 form a conduit from H263 in the active site to the protein exterior and function in proton extraction from the porphyrin macrocycle. The role of H263 as the porphyrin proton-accepting residue is central to catalysis since metalation only occurs in conjunction with proton abstraction. It is suggested that iron is transported from the exterior of the enzyme at D383/H231 via residues W227 and Y191 to the site of metalation at residues R164 and Y165 which are on the opposite side of the active site pocket from H263. This model should be general for mitochondrial membrane-associated eucaryotic ferrochelatases but may differ for bacterial ferrochelatases since the spatial orientation of the enzyme within prokaryotic cells may differ.     

Directed mutagenesis of apecific active site residues on Fibrobacter succinogenes 1,3-1,4-beta -D-glucanase significantly affects catalysis and enzyme structural stability

The functional and structural significance of amino acid residues Met(39), Glu(56), Asp(58), Glu(60), and Gly(63) of Fibrobacter succinogenes 1,3-1,4-beta-d-glucanase was explored by the approach of site-directed mutagenesis, initial rate kinetics, fluorescence spectroscopy, and CD spectrometry. Glu(56), Asp(58), Glu(60), and Gly(63) residues are conserved among known primary sequences of the bacterial and fungal enzymes. Kinetic analyses revealed that 240-, 540-, 570-, and 880-fold decreases in k(cat) were observed for the E56D, E60D, D58N, and D58E mutant enzymes, respectively, with a similar substrate affinity relative to the wild type enzyme. In contrast, no detectable enzymatic activity was observed for the E56A, E56Q, D58A, E60A, and E60Q mutants. These results indicated that the carboxyl side chain at positions 56 and 60 is mandatory for enzyme catalysis. M39F, unlike the other mutants, exhibited a 5-fold increase in K(m) value. Lower thermostability was found with the G63A mutant when compared with wild type or other mutant forms of F. succinogenes 1,3-1,4-beta-d-glucanase. Denatured wild type and mutant enzymes were, however, recoverable as active enzymes when 8 m urea was employed as the denaturant. Structural modeling and kinetic studies suggest that Glu(56), Asp(58), and Glu(60) residues apparently play important role(s) in the catalysis of F. succinogenes 1,3-1,4-beta-d-glucanase.     

Carboxyl-terminal hydrophilic tail of a NhaP type Na+/H+ antiporter from cyanobacteria is involved in the apparent affinity for Na+ and pH sensitivity

Little information is available on the C-terminal hydrophilic tails of prokaryotic Na(+)/H(+) antiporters. To address functional properties of the C-terminal tail, truncation mutants in this domain were constructed. Truncation of C-terminal amino acid residues of NhaP1 type antiporter from Synechocystis PCC6803 (SynNhaP1) did not change the V(max) values, but increased the K(m) values for Na(+) and Li(+) about 3 to 15-fold. Truncation of C-terminal tail of a halotolerant cyanobacterium Aphanothece halophytica (ApNhaP1) significantly decreased the V(max) although it did not alter the K(m) values for Na(+). The C-terminal part of SynNhaP1 was expressed in E. coli and purified as a 16kDa soluble protein. Addition of purified polypeptide to the membrane vesicles expressing the C-terminal truncated SynNhaP1 increased the exchange activities. Change of Glu519 and Glu521 to Lys in C-terminal tail altered the pH dependence of Na(+)/H(+) and Li(+)/H(+) exchange activities. These results indicate that the specific acidic amino acid residues at C-terminal domain play important roles for the K(m) and the pH dependence of the exchange activity.     

Thr-774 (transmembrane segment M5), Val-920 (M8), and Glu-954 (M9) are involved in Na+ transport, and Gln-923 (M8) is essential for Na,K-ATPase activity

The highly conserved amino acids of rat Na,K-ATPase, Thr-774 in the transmembrane helices M5, Val-920 and Gln-923 in M8, and Glu-953 and Glu-954 in M9, the side chains of which appear to be in close proximity, were mutated, and the resulting proteins, T774A, E953A/K, and E954A/K, V920E and Q923N/E/D/L, were expressed in HeLa cells. Ouabain-resistant cell lines were obtained from T774A, V920E, E953A, and E954A, whereas Q923N/E/D/L, E953K, and E954K could only be transiently expressed as fusion proteins with an enhanced green fluorescent protein. The apparent K0.5 values for Na+, as estimated by the Na+-dependent phosphoenzyme formation (K0.5(Na,EP)) or Na,K-ATPase activity (K(0.5)(Na,ATPase)), were increased by around 2 approximately 8-fold in the case of T774A, V920E, and E954A. The apparent K0.5 values for K+, as estimated by the Na,K-ATPase (K0.5(K,ATPase)) or p-nitrophenylphosphatase activity (K0.5(K,pNPPase)), were affected only slightly by the 3 mutations, except that V920E showed a 1.7-fold increase in the K0.5(K,ATPase). The apparent K0.5 values for ATP (K0.5(EP)), as estimated by phosphorylation (a high affinity ATP effect), were increased by 1.6 approximately 2.6-fold in the case of T774A, V920E, and E954A. Those estimated by Na,K-ATPase activity (K0.5(ATPase)) and ATP-induced inhibition (K(i,0.5)(pNPPase)) of K-pNPPase activity (low affinity ATP effects) were, respectively, increased by 1.8-fold and unchanged in the case of T774A but decreased by 2- and 4.8-fold in the case of V920E and were slightly changed and increased by 1.7-fold in the case of E954A. The E953A showed little significant change in the apparent affinities. These results suggest that Gln-923 in M8 is crucial for the active transport of Na+ and/or K+ across membranes and that the side chain oxygen atom of Thr-774 in M5, the methyl group(s) of Val-920 in M8, and the carboxyl oxygen(s) of Glu-954 in M9 mainly play some role in the transport of Na+ and also in the high and low affinity ATP effects rather than the transport of K+.     

Stabilization of the actomyosin complex by negative charges on myosin

Sequence comparisons of members of the myosin superfamily show a high degree of charge conservation in a surface exposed helix (Dictyostelium discoideum myosin II heavy chain residues S510 to K546). Most myosins display a triplet of acidic residues at the equivalent positions to D. discoideummyosin II residues D530, E531, and Q532. The high degree of charge conservation suggests strong evolutionary constrain and that this region is important for myosin function. Mutations at position E531 were shown to strongly affect actin binding [Giese, K. C., and Spudich, J. A. (1997) Biochemistry 36, 8465-8473]. Here, we used steady-state and transient kinetics to characterize the enzymatic competence of mutant constructs E531Q and Q532E, and their properties were compared with those of a loop 2 mutant with a 20 amino acid insertion containing 12 positive charges (20/+12) [Furch et al. (1998) Biochemistry 37, 6317-6326], double mutant Q532E(20/+12), and the native motor domain constructs. Our results confirm that charge changes at residues 531 and 532 primarily affect actin binding with little change being communicated to the nucleotide pocket. Mutation D531Q reduces actin affinity (K(A)) 10-fold, while Q532E leads to a 5-fold increase. The observed changes in K(A)() stem almost exclusively from variations in the dissociation rate constant (k(-A)), with the introduction of a single negative charge at position 532 having the same effect on k(-A) as the introduction of 12 positive charges in the loop 2 region.     

A conserved negatively charged cluster in the active site of creatine kinase is critical for enzymatic activity

Creatine kinase catalyzes the reversible transphosphorylation of creatine by MgATP. From the sequence homology and the molecular structure of creatine kinase isoenzymes, we have identified several highly conserved residues with a potential function in the active site: a negatively charged cluster (Glu(226), Glu(227), Asp(228)) and a serine (Ser(280)). Mutant proteins E226Q, E226L, E227Q, E227L, D228N, and S280A/S280D of human sarcomeric mitochondrial creatine kinase were generated by in vitro mutagenesis, expressed in Escherichia coli, and purified to homogeneity. Their overall structural integrity was confirmed by CD spectroscopy and gel filtration chromatography. The enzymatic activity of all proteins mutated in the negatively charged cluster was extremely low (0.002-0.4% of wild type) and showed apparent Michaelis constants (K(m)) similar to wild type, suggesting that most of the residual activity may be attributed to wild-type revertants. Mutations of Ser(280) led to higher residual activities and altered K(m) values; S280A showed an increase of K(m) for phosphocreatine (65-fold), creatine (6-fold), and ATP (6-fold); S280D showed a decrease of K(m) for creatine (6-fold). These results, together with the transition state structure of the homologous arginine kinase (Zhou, G., Somasundaram, T., Blanc, E., Parthasarathy G., Ellington, W. R., and Chapman, M. S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8449-8454), strongly suggest a critical role of Glu(226), Glu(227), and Asp(228) in substrate binding and catalysis and point to Glu(227) as a catalytic base.     

Structure-based engineering of Alcaligenes xylosoxidans copper-containing nitrite reductase enhances intermolecular electron transfer reaction with pseudoazurin

The intermolecular electron transfer from Achromobacter cycloclastes pseudoazurin (AcPAZ) to wild-type and mutant Alcaligenes xylosoxidans nitrite reductases (AxNIRs) was investigated using steady-state kinetics and electrochemical methods. The affinity and the electron transfer reaction constant (k(ET)) are considerably lower between AcPAZ and AxNIR (K(m) = 1.34 mM and k(ET) = 0.87 x 10(5) M(-1) s(-1)) than between AcPAZ and its cognate nitrite reductase (AcNIR) (K(m) = 20 microM and k(ET) = 7.3 x 10(5) M(-1) s(-1)). A negatively charged hydrophobic patch, comprising seven acidic residues around the type 1 copper site in AcNIR, is the site of protein-protein interaction with a positively charged hydrophobic patch on AcPAZ. In AxNIR, four of the negatively charged residues (Glu-112, Glu-133, Glu-195, and Asp-199) are conserved at the corresponding positions of AcNIR, whereas the other three residues are not acidic amino acids but neutral amino acids (Ala-83, Ala-191, and Gly-198). Seven mutant AxNIRs with additional negatively charged residues surrounding the hydrophobic patch of AxNIR (A83D, A191E, G198E, A83D/A191E, A93D/G198E, A191E/G198E, and A83D/A191E/G198E) were prepared to enhance the specificity of the electron transport reaction between AcPAZ and AxNIR. The k(ET) values of these mutants become progressively larger as the number of mutated residues increases. The K(m) and k(ET) values of A83D/A191E/G198E (K(m) = 88 microM and k(ET) = 4.1 x 10(5) M(-1) s(-1)) are 15-fold smaller and 4.7-fold larger than those of wild-type AxNIR, respectively. These results suggest that the introduction of negatively charged residues into the docking surface of AxNIR facilitates both the formation of electron transport complex and the electron transfer reaction.