Functional consequences of alterations to Gly310, Gly770, and Gly801 located in the transmembrane domain of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Site-specific mutagenesis was used to replace Gly310, Gly770, and Gly801, located in the transmembrane domain of the sarcoplasmic reticulum Ca(2+)-ATPase, with either alanine or valine. In addition, Gly310 was substituted with proline. In the Gly310----Ala mutant, the Vmax for Ca2+ transport and ATPase activity was reduced to about 40% of the wild type activity, but the apparent Ca2+ affinity was close to normal. The Gly310----Val and Gly310----Pro mutants were devoid of Ca2+ transport or ATPase activity and displayed more than a 20-fold reduction in the apparent Ca2+ affinities measured in the phosphorylation assays with either ATP or Pi. In these mutants, the rate of phosphoenzyme hydrolysis was reduced, and the ADP-insensitive phosphoenzyme intermediate accumulated. The apparent affinity for Pi was increased in the absence, but not in the presence, of dimethyl sulfoxide. The properties of this new class of Ca(2+)-ATPase mutants ("E2/E2P" type) are consistent with a conformational state in which the protein-phosphate interaction is stabilized and the Ca(2+)-protein interaction is destabilized. The Gly770----Ala mutant transported Ca2+ with a Vmax close to that of the wild type, but displayed more than a 20-fold reduction of apparent Ca2+ affinity. The Gly770----Val mutant was not phosphorylated from either ATP or Pi. The Gly801----Ala mutant transported Ca2+ with a Vmax of 126% that of the wild type, hydrolyzed ATP at the same Vmax as the wild type in the presence of calcium ionophore, and displayed a 3-fold reduction in apparent Ca2+ affinity. The Gly801----Val mutant was unable to transport Ca2+ and to be phosphorylated from ATP, even at a Ca2+ concentration of 1 mM, but Ca2+ in the micromolar range inhibited phosphorylation from Pi. The ability to bind ATP with normal affinity was retained. The properties of this mutant are consistent with a disruption of one of the two Ca2+ binding sites required for phosphorylation with ATP.     

Isolation and characterization of an analogue-resistant aminoacyl-tRNA synthetase mutant in Aspergillus nidulans.

Six mutants resistant to p-fluorophenylalanine (FPA) were selected on a medium containing aspartate as the sole source of nitrogen using a phenylalanine-requiring (phenA)auxotroph of A. nidulans as the wild type. The mutants, on the basis of genetic characterization, were found to be alleilic and located on the left arm of the linkage group III, approximately 13 map unit left to meth H locus, henceforth assigned to the symbol fpaV. At a fixed concentration of phenylalanine (23 micrograms/ml), the LD50 value of FPA for all the six mutants was found to be about three times more than that for the wild type strain. Affinity chromatographic purification of the enzyme phenylalanyl-tRNA (Phe-tRNA) synthetase from the mutant as well as the wild type strains, revealed that the wild type enzyme had about 1.4-fold higher affinity for phenylalanine as compared to that for FPA, both in the affinity column and in the catalytic reaction. However, the mutant enzyme showed almost a similar affinity for both in columns but a greatly reduced affinity for FPA in the catalytic reaction.     

Altered prephenate dehydratase in phenylalanine-excreting mutants of Brevibacterium flavum.

The regulatory properties of three key enzymes in the phenylalanine biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase (DAHP synthetase) [EC 4.1.2.15], chorismate mutase [EC 5.4.99.5], and prephenate dehydratase [prephenate hydro-lyase (decarboxylating), EC 4.2.1.51] were compared in three phenylalanine-excreting mutants and the wild strain of Brevibacterium flavum. Regulation of DAHP synthetase by phenylalanine and tyrosine in these mutants did not change at all, but the specific activities of the mutant cell extracts increased 1.3- to 2.8-fold, as reported previously (1). Chorismate mutase activities in both the wild and the mutant strains were cumulatively inhibited by phenylalanine and tyrosine and recovered with tryptophan, while the specific activities of the mutants increased 1.3- to 2.8-fold, like those of DAHP synthetase. On the other hand, the specific activities of prephenate dehydratase in the mutant and wild strains were similar, when tyrosine was present. While prephenate dehydratase of the wild strain was inhibited by phenylalanine, tryptophan, and several phenylalanine analogues, the mutant enzymes were not inhibited at all but were activated by these effectors. Tyrosine activated the mutant enzymes much more strongly than the wild-type enzyme: in mutant 221-43, 1 mM tyrosine caused 28-fold activation. Km and the activation constant for tyrosine were slightly altered to a half and 6-fold compared with the wild-type enzyme, respectively, while the activation constants for phenylalanine and tryptophan were 500-fold higher than the respective inhibition constants of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 x 10(5), a half of that of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 X 10(5) a half of that of the wild type enzyme, while in the presence of tyrosine, phenylalanine, or tryptophan, it increased to that of the wild-type enzyme. Immediately after the mutant enzyme had been activated by tyrosine and then the tyrosine removed, it still showed about 10-fold higher specific activity than before the activation by tyrosine. However, on standing in ice the activity gradually fell to the initial level before the activation by tyrosine. Ammonium sulfate promoted the decrease of the activity. On the basis of these results, regulatory mechanisms for phenylalanine biosynthesis in vivo as well as mechanisms for the phenylalanine overproduction in the mutants are discussed.     

Single-turnover analysis of mutant human apurinic/apyrimidinic endonuclease

Apurinic/apyrimidinic endonuclease (AP endo) is a key enzyme in the repair of oxidatively damaged DNA. Using single-turnover conditions, we recently described substrate binding parameters for wild type human AP endo. In this study, we utilized four enzyme mutants, D283A, D308A, D283A/D308A, and H309N, and assayed them under steady state and single-turnover conditions. The turnover number of the single aspartate mutants was decreased 10-30-fold in comparison to that of the wild type. The decrease in the turnover number was accompanied by a 17- and 50-fold decrease in the forward rate constant (kon) for substrate binding by D308A and D283A, respectively. The dissociation rate constant for substrate (koff) was unchanged for the D308A mutant but was 10 times faster for the D283A mutant than for the wild type. The apparent Km values for both of the single aspartate mutants were about equal to their respective KD values. To account for the kinetic behavior of the D308A mutant, it was necessary to insert a conformational change into the kinetic scheme. In contrast to the single aspartate mutants, the turnover number for the double mutant was 500-fold lower than that of the wild type, its apparent Km was 2.5-fold higher, and binding to substrate was weak. Mutation of His309 caused the greatest decrease in activity, resulting in a turnover number that was more than 30000-fold lower than that of the wild type and an apparent Km that was 13-fold higher, supporting the notion that His309 is intimately involved in catalysis. Molecular dynamics simulation techniques suggested that conversion of either aspartate to alanine resulted in major shifts in the spatial localization of key amino acids. Despite the fact that the two aspartates flank His309, the movement they engendered was distinct, consistent with the differences in catalytic behavior. We suggest that the conformation of the active site is largely maintained by the two aspartates, which enable efficient binding and cleavage of abasic site-containing DNA.     

Functional consequences of alterations to Pro328 and Leu332 located in the 4th transmembrane segment of the alpha-subunit of the rat kidney Na+,K(+)-ATPase.

Site-specific mutagenesis was used to analyse the functional roles of the residues Pro328 and Leu332 located in the conserved PEGLL motif of the predicted transmembrane helix M4 in the alpha 1-subunit of the ouabain resistant rat kidney Na+,K(+)-ATPase. cDNAs encoding either of the Na+,K(+)-ATPase mutants Pro328-->Ala and Leu332-->Ala, and wild type, were cloned into the expression vector pMT2 and transfected into COS-1 cells. Ouabain-resistant clones growing in the presence of 10 microM ouabain were isolated, and the Na+,K+, ATP and pH dependencies of the Na+,K(+)-ATPase activity measured in the presence of 10 microM ouabain were analysed. Under these conditions the exogenous expressed Na+,K(+)-ATPase contributed more than 95% of the Na+,K(+)-ATPase activity. The Pro328-->Ala mutant displayed a reduced apparent affinity for Na+ (K0.5 (Na+) 13.04 mM), relative to the wild type (K0.5 (Na+) 7.13 mM). By contrast, the apparent affinity for Na+ displayed by the Leu332-->Ala mutant was increased (K0.5 (Na+) 3.92 mM). Either of the mutants exhibited lower apparent affinity for K+ relative to the wild type (K0.5 (K+) 2.46 mM for Pro328-->Ala and 1.97 mM for Leu332-->Ala, compared with 0.78 mM for wild type). Both mutants exhibited higher apparent affinity for ATP than the wild type (K0.5 (ATP) 0.086 mM for Pro328-->Ala and 0.042 mM for Leu332-->Ala, compared with 0.287 mM for wild type). The influence of pH was in accordance with an acceleration of the E2 (K)-->E1 transition in the mutants relative to the wild type. These data are consistent with a role of Pro328 and Leu332 in the stabilization of the E2 form and of Pro328 in Na+ binding. The possible role of the mutated residues in K+ binding is discussed.     

Mutation of active site residues of insulin-degrading enzyme alters allosteric interactions

The active site glutamate (Glu(111)) and the active site histidine (His(112)) of insulin-degrading enzyme (IDE) were mutated. These mutant enzymes exhibit, in addition to a large decrease in catalytic activity, a change in the substrate-velocity response from a sigmoidal one seen with the native enzyme (Hill coefficient > 2), to a hyperbolic response. With 2-aminobenzoyl-GGFLRKHGQ-N-(2,4-dinitrophenyl)ethylenediamine as substrate, ATP and triphosphate increase the reaction rate of the wild type enzyme some 50-80-fold. This effect is dampened with glutamate mutants to no effect or less than a 3-fold increase in activity and changed to inhibition with the histidine mutants. Sedimentation equilibrium shows the IDE mutants exhibit a similar oligomeric distribution as the wild type enzyme, being predominantly monomeric, with triphosphate having little if any effect on the oligomeric state. Triphosphate did induce aggregation of many of the IDE mutants. Thus, the oligomeric state of IDE does not correlate with kinetic properties. The His(112) mutants were shown to bind zinc, but with a lower affinity than the wild type enzyme. The glutamate mutants displayed an altered cleavage profile for the peptide beta-endorphin. Wild type IDE cleaved beta-endorphin at Leu(17)-Phe(18) and Phe(18)-Lys(19), whereas the glutamate mutants cleaved at these sites, but in addition at Lys(19)-Asn(20) and at Met(5)-Thr(6). Thus, active site mutations of IDE are suggested to not only reduce catalytic activity but also cause local conformational changes that affect the allosteric properties of the enzyme.     

Phosphorylation of human CTP synthetase 1 by protein kinase A: identification of Thr455 as a major site of phosphorylation

CTP synthetase is an essential enzyme that generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this study, we examined the phosphorylation of the human CTPS1-encoded CTP synthetase 1 by protein kinase A. CTP synthetase 1 was expressed and purified from a Saccharomyces cerevisiae ura7Delta ura8Delta double mutant that lacks CTP synthetase activity. Using purified CTP synthetase 1 as a substrate, protein kinase A activity was time- and dose-dependent. The phosphorylation, which primarily occurred on a threonine residue, was accompanied by a 50% decrease in CTP synthetase 1 activity. The synthetic peptide LGKRRTLFQT that contains the protein kinase A motif for Thr(455) was a substrate for protein kinase A. A Thr(455) to Ala (T455A) mutation in CTP synthetase 1 was constructed by site-directed mutagenesis and was expressed and purified from the S. cerevisiae ura7Delta ura8Delta mutant. The T455A mutation caused a 78% decrease in protein kinase A phosphorylation and the loss of the phosphothreonine residue and a major phosphopeptide that were present in the purified wild type enzyme phosphorylated by protein kinase A. The CTP synthetase 1 activity of the T455A mutant enzyme was 2-fold higher than the wild type enzyme. In addition, the T455A mutation caused a 44% decrease in the amount of human CTP synthetase 1 that was phosphorylated in S. cerevisiae cells, and this was accompanied by a 2.5-fold increase in the cellular concentration of CTP and a 1.5-fold increase in the choline-dependent synthesis of phosphatidylcholine.     

Designing an allosterically locked phosphofructokinase

Six site-directed mutants of Escherichia coli phosphofructokinase (PFK) were made in an attempt to produce an enzyme "locked" in the inactive or "T"-state. The kinetic properties of the mutants were examined as a function of the substrates fructose 6-phosphate (Fru6P) and ATP, the positive effector GDP, and the negative effector phosphoenolpyruvate (PEP). All mutants exhibited lower activity than wild-type PFK. Three mutants (RS63, LV153, and VT246) had apparent dissociation constants for substrates and effectors similar to those of wild type. One mutant, HN160, had a 10-fold reduced affinity for Fru6P and reduced apparent affinity for the effectors. Two mutants, SN159 and T(GS)156, exhibited hyperbolic kinetics consistent with a "locked" T-state protein. Surprisingly, T(GS)156 showed hyperbolic activation in response to the physiological inhibitor PEP. The mutant PFK properties are discussed in terms of the PFK structure. These results suggest that the kinetic properties of PFK are sensitive to interactions in the homotropic interface; residues 156-160 in particular are critical in mediating the interactions between effector and active sites and in the T to R quaternary transition.     

Identification of residues of Escherichia coli phosphofructokinase that contribute to nucleotide binding and specificity

The apparent affinity of phosphofructo-1-kinase (PFK) of Escherichia coli for ATP is at least 10 times higher than for other nucleotides. Mutagenesis was directed toward five residues that may interact with ATP: Y41, F76, R77, R82, and R111. Alanine at position 41 or 76 increased the apparent Km by 49- and 62-fold, respectively. Position 41 requires the presence of a large hydrophobic residue and is not restricted to aromatic rings. Tryptophan and, to a lesser extent, phenylalanine could substitute at position 76. None of the mutants at 41 or 76 showed a change in the preference for alternative purines, although F76W used CTP 3 times better than the wild type enzyme. Mutations of R77 suggested that the interaction was hydrophobic with no influence on nucleotide preference. Mutation of R82 to alanine or glutamic acid increased the apparent Km for ATP by more than 20-fold and lowered the kcat/Km with ATP more than 30-fold. However, these mutants had a higher kcat/Km than wild type for both GTP and CTP, reflecting a loss of substrate preference. A loss in preference is seen as well with R111A where the kcat/Km for ATP decreases by only 68%, but the kcat/Km with GTP increases more than 10-fold. Activities with ITP, CTP, and UTP are also higher than with the wild type enzyme. Arginine residues at positions 82 and 111 are important dictators of nucleoside triphosphate preference.     

Characterization of single step albizziin-resistant Chinese hamster ovary cell lines with elevated levels of asparagine synthetase activity

The amino acid analog, albizziin, which acts as a competitive inhibitor of asparagine synthetase with respect to glutamine was used to isolate mutants of Chinese hamster ovary cells with alterations in levels of the target enzyme. These mutant lines have been characterized biochemically and genetically. Mutants selected in a single step are up to 40-fold more resistant to the drug than the parental line, express levels of asparagine synthetase activity 6-17-fold greater than that of wild type cells, and act co-dominantly in hybrids. Several classes of mutations can be distinguished on the basis of cross-resistance to beta-aspartyl hydroxamate, another amino acid analog. Studies on asparagine synthetase indicate that resistance to albizziin may be due to altered regulation of asparagine synthetase, structural mutations of the enzyme, and gene amplification.     

Biosynthesis of bacterial glycogen. Use of site-directed mutagenesis to probe the role of tyrosine 114 in the catalytic mechanism of ADP-glucose synthetase from Escherichia coli

Previous covalent modification studies showed that tyrosine 114 of Escherichia coli ADP-glucose synthetase is involved in substrate binding (Lee, Y. M., and Preiss, J. (1986) J. Biol. Chem. 261, 1058-1064). We have prepared, via site-directed mutagenesis, an E. coli ADP-glucose synthetase variant (Phe114) containing a Tyr114 to Phe substitution in order to test whether the phenolic hydroxyl group plays a critical role in catalysis. Kinetic characterization of Phe114 ADP-glucose synthetase indicates that the Tyr114 hydroxyl is not obligatory for the enzyme catalysis. However, the variant enzyme showed altered properties. It showed a decreased apparent affinity for the substrates. The variant enzyme showed less than 2-fold activation by 5 mM fructose 1,6-bisphosphate in the ADP-glucose synthesis direction. In contrast, in the pyrophosphorolysis direction, the mutant enzyme showed about a 30-fold activation by 5 mM fructose 1,6-bisphosphate. The variant enzyme is heat-labile compared to wild type enzyme. It lost about 60% enzyme activity on incubation at 65 degrees C for 5 min in the presence of 30 mM Pi. The wild type enzyme is stable under these conditions. The results indicate that tyrosine 114 is involved directly or indirectly in enzyme catalysis, but is not obligatory for the enzyme catalysis. Conversion of Tyr114 to Phe also alters the regulatory properties of the enzyme with respect to activation by fructose-1,6-P2 and inhibition by AMP.     

Mutagenesis of the folC gene encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli

The folC gene of Escherichia coli, cloned in a pUC19 vector, was mutagenized by progressive deletions from both the 5' and the 3' ends and by TAB linker insertion. A number of 5'-deleted genes, which had the initiator ATG codon removed, produced a truncated gene product, in reduced amounts, from a secondary initiation site. The most likely position of this site at a GTG codon located 35 codons downstream of the normal start site. This product could complement the folC mutation in E. coli strain SF4 as well as a strain deleted in the folC gene. The specific activity of extracts of the mutant enzyme are 4-16% that of the wild type enzyme for the folylpolyglutamate synthetase activity and 6-19% for the dihydrofolate synthetase activity. The relative amount of protein expressed by the mutant, compared to the wild type, in maxicells was comparable to the relative specific activity, suggesting that the kcat of the mutant enzyme is similar to that of the wild type. Mutants with up to 14 amino acids deleted from the carboxy terminal could still complement the folC deletion mutant. Seven out of ten linker insertions dispersed through the coding region of the gene showed complementation of the folC mutation in strain SF4 but none of these insertion mutants were able to complement the strain containing a deleted folC gene. None of the carboxy terminal or linker insertion mutants had a specific activity greater than 0.5% that of the wild type enzyme. The dihydrofolate synthetase and folylpolyglutamate synthetase activities behaved similarly in all mutants, both retaining a large fraction of the wild type activity in the amino terminal deletions and both being very low in the carboxy terminal deletions and linker insertion mutants. These studies are consistent with a single catalytic site for the two activities catalyzed by this enzyme.     

Dissection of the functional differences between sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 1 and 2 isoforms and characterization of Darier disease (SERCA2) mutants by steady-state and transient kinetic analyses

Steady-state and rapid kinetic studies were conducted to functionally characterize the overall and partial reactions of the Ca2+ transport cycle mediated by the human sarco(endo)plasmic reticulum Ca2+-ATPase 2 (SERCA2) isoforms, SERCA2a and SERCA2b, and 10 Darier disease (DD) mutants upon heterologous expression in HEK-293 cells. SERCA2b displayed a 10-fold decrease in the rate of Ca2+ dissociation from E1Ca2 relative to SERCA2a (i.e. SERCA2b enzyme manifests true high affinity at cytosolic Ca2+ sites) and a lower rate of dephosphorylation. These fundamental kinetic differences explain the increased apparent affinity for activation by cytosolic Ca2+ and the reduced catalytic turnover rate in SERCA2b. Relative to SERCA1a, both SERCA2 isoforms displayed a 2-fold decrease of the rate of E2 to E1Ca2 transition. Furthermore, seven DD mutants were expressed at similar levels as wild type. The expression level was 2-fold reduced for Gly23 --> Glu and Ser920 --> Tyr and 10-fold reduced for Gly749 --> Arg. Uncoupling between Ca2+ translocation and ATP hydrolysis and/or changes in the rates of partial reactions account for lack of function for 7 of 10 mutants: Gly23 --> Glu (uncoupling), Ser186 --> Phe, Pro602 --> Leu, and Asp702 --> Asn (block of E1 approximately P(Ca2) to E2-P transition), Cys318 --> Arg (uncoupling and 3-fold reduction of E2-P to E2 transition rate), and Thr357 --> Lys and Gly769 --> Arg (lack of phosphorylation). A 2-fold decrease in the E1 approximately P(Ca2) to E2-P transition rate is responsible for the 2-fold decrease in activity for Pro895 --> Leu. Ser920 --> Tyr is a unique DD mutant showing an enhanced molecular Ca2+ transport activity relative to wild-type SERCA2b. In this case, the disease may be a consequence of the low expression level and/or reduction of Ca2+ affinity and sensitivity to inhibition by lumenal Ca2+.     

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.     

L-Methionine SR-sulfoximine-resistant glutamine synthetase from mutants of Salmonella typhimurium

Two mutants of Salmonella typhimurium resistant to growth inhibition by the glutamine synthetase transition state analog, L-methionine SR-sulfoximine, were isolated and characterized. These mutants are glutamine bradytrophs and cannot use growth rate-limiting nitrogen sources. Although this phenotype resembles that of mutants with lesions in the regulatory gene for glutamine synthetase, glnG, these mutations do not lie in the glnG gene. Purification and characterization of the glutamine synthetase from one of the mutants and a control strain demonstrated that the mutant enzyme is defective in the reverse gamma-glutamyltransferase activity but has biosynthetic activity that is resistant to inhibition by L-methionine SR-sulfoximine. The mutant enzyme also has a 4.4-fold higher apparent Km for glutamate (0.2 mM versus 2.1 mM, respectively) and a 13.8-fold higher Km for NH3 (6.4 mM versus 0.46 mM) than the enzyme from the control. These data show that the glutamine synthetase protein has been altered by this mutation, designated as glnA982, and suggest that the L-methionine SR-sulfoximine resistance is conferred by a change in the NH3 binding domain of the enzyme.     

(Na, K)ATPase activity in membrane preparations of ouabain-resistant HeLa cells.

Membrane preparations from two independent ouabain-resistant HeLa cell clones, HI-B1 and HI-C1, each appear to contain two species of (Na,K)ATPase. Two-thirds of the total (Na,K)ATPase in each mutant is indistinguishable from the enzyme in preparations of wild type cells with respect to ouabain binding, ouabain inhibition of (Na,K)ATPase activity, and dependence of ATP hydrolysis on Na, Mg, K, and ATP concentration. The remaining (Na,K)ATPase activity in the mutants is up to 1000 and 10 000 times, respectively, more resistant to ouabain than wild type enzyme. Resistance results from a lower affinity of the mutant enzymes for the inhibitor. The presence of Na, K, or Mg has little or no effect on the degree of resistance expressed by the mutant enzymes, although the resistance of the wild type enzyme varies 400-fold in the presence of different ligands. Incubation with 5 X 10(-8) M ouabain abolishes the activity of the wild type enzyme without affecting the activity of the resistant enzymes. Using this procedure we compared the parameters of ATP hydrolysis via the resistant and wild type enzymes. Ouabain-resistant (Na,K)ATPase of HI-C1 has an apparent K0.5 for potassium 3-4 times higher than that of either wild type enzyme or the resistant enzyme of HI-B1.     

Glutamine synthetase in ribulose 1,5-bisphosphate carboxylase/oxygenase deficient tobacco mutants in cell suspension culture

In two tobacco mutants lacking ribulose, 1,5-bisphosphate carboxylase/oxygenase the amount of glutamine synthetase and its activity were determined and compared with the wild type green cells. It was shown that in these two mutants glutamine synthetase protein content was six times lower than in the wild type. This situation was comparable to that found in etiolated cells where ribulose 1,5-bisphosphate carboxylase/oxygenase was absent. These observations suggest that a common regulatory mechanism might control the dual light dependent biosynthesis of both enzymes. The results have also implications concerning the efficiency of the reassimilation of ammonia by chloroplastic glutamine synthetase during the photorespiratory process.     

Altered metabolism of thrombospondin by Chinese hamster ovary cells defective in glycosaminoglycan synthesis

We examined the ability of Chinese hamster ovary (CHO) cell mutants defective in glycosaminoglycan synthesis to metabolize 125I-labeled thrombospondin (TSP). Wild type CHO cells bound and degraded 125I-TSP with kinetics similar to those reported for endothelial cells. Both binding and degradation were saturable (half-saturation at 20 micrograms/ml). When the concentration of labeled TSP was 1-5 micrograms/ml, mutant 745, defective in xylosyltransferase, and mutant 761, defective in galactosyltransferase I, bound and degraded 6- to 16-fold less TSP than wild type; mutant 803, which specifically lacks heparan sulfate chains, bound and degraded 5-fold less TSP than wild type; and mutant 677, which lacks heparan sulfate and has increased levels of chondroitin sulfate, bound and degraded 2-fold less TSP than wild type. Binding and degradation of TSP by the mutants were not saturable at TSP concentrations up to 100 micrograms/ml. Bound TSP was localized by immunofluorescence to punctate structures on wild type and, to a lesser extent, 677 cells. Heparitinase pretreatment of wild type cells caused a 2- to 3-fold decrease in binding and degradation, whereas chondroitinase pretreatment had no effect. Chondroitinase pretreatment of the 677 mutant (deficient heparan sulfate and excess chondroitin sulfate) caused a 2-fold decrease in binding and an 8-fold decrease in turnover, whereas heparitinase pretreatment had no effect. Treatment of wild type cells with both heparitinase and chondroitinase resulted in a 6- to 8-fold decrease in binding and turnover. These results indicate that cell surface proteoglycans mediate metabolism of TSP by CHO cells and that the primary effectors of TSP metabolism are heparan sulfate proteoglycans.     

Site-directed mutagenesis of the putative distal helix of peroxygenase cytochrome P450

CYP152A1 is an unusual, peroxygenase enzyme that catalyzes the beta- or alpha-hydroxylation of fatty acids by efficiently introducing an oxygen atom from H2O2 to the fatty acid. To clarify the mechanistic roles of amino acid residues in this enzyme, we have used site-directed mutagenesis of residues in the putative distal helix and measured the spectroscopic and enzymatic properties of the mutant proteins. Initially, we carried out Lys-scanning mutagenesis of amino acids in this region to determine residues of CYP152A1 that might have a mechanistic role. Among the Lys mutants, only P243K gave an absorption spectrum characteristic of a nitrogenous ligand-bound form of a ferric P450. Further investigation of the Pro243 site revealed that a P243H mutant also exhibited a nitrogen-bound form, but that the mutants P243A or P243S did not. On the hydroxylation of myristic acid by the Lys mutants, we observed a large decrease in activity for R242K and A246K. We therefore examined other mutants at amino acid positions 242 and 246. At position 246, an A246K mutant showed a roughly 19-fold lower affinity for myristic acid than the wild type. Replacing Ala246 with Ser decreased the catalytic activity, but did not affect affinity for the substrate. An A246V mutant showed slightly reduced activity and moderately reduced affinity. At position 242, an R242A showed about a fivefold lower affinity than the wild type for myristic acid. The Km values for H2O2 increased and Vmax values decreased in the order of wild type, R242K, and R242A when H2O2 was used; furthermore, Vmax/Km was greatly reduced in R242A compared with the wild type. If cumene hydroperoxide was used instead of H2O2, however, the Km values were not affected much by these substitutions. Together, our results suggest that in CYP152A1 the side chain of Pro243 is located close to the iron at the distal side of a heme molecule; the fatty acid substrate may be positioned near to Ala246 in the catalytic pocket, although Ala246 does not participate in hydrophobic interactions with the substrate; and that Arg242 is a critical residue for substrate binding and H2O2-specific catalysis.     

The amino acid sequence 442GDASE446 in Na/K-ATPase is an important motif in forming the high and low affinity ATP binding pockets

A highly conserved amino acid sequence 442GDASE446 in the ATP binding pocket of rat Na/K-ATPase was mutated, and the resulting proteins, G442A, G442P, D443A, S445A, and E446A, were expressed in HeLa cells to investigate the effect of individual ligands on Na/K-ATPase. The apparent Km for the high and low affinity ATP effects was estimated by ATP concentration dependence for the formation of the Na-dependent phosphoenzyme (Kmh) and Na/K-ATPase activity (Kml). The apparent Km for p-nitrophenylphosphate (pNPP) for K-dependent-pNPPase (KmP) and its inhibition by ATP (Ki,0.5) and the apparent Km for Mg2+, Na+, K+, and vanadate in Na/K-ATPase were also estimated. For all the mutants, the value for ATP was approximately 2-10-fold larger than that of the wild type. While the turnover number for Na/K-ATPase activity were unaffected or reduced by 20 approximately 50% in mutants G442(A/P) and D443A. Although both affinities for ATP effects were reduced as a result of the mutations, the ratio, Kml Kmh, for each mutant was 1.3 approximately 3.7, indicating that these mutations had a greater impact on the low affinity ATP effect than on the high affinity effect. Each KmP value with the turnover number suggests that these mutations favor the binding of pNPP over that of ATP. These data and others indicate that the sequence 442GDASE446 in the ATP binding pocket is an important motif that it is involved in both the high and low affinity ATP effects rather than in free Mg2+, Na+, and K+ effects.