Participation of ADP dissociation in the rate-determining step in cAMP-dependent protein kinase

Pre-steady-state kinetic analyses of the catalytic subunit of cAMP-dependent protein kinase showed that the rate constant for phosphoryl transfer is fast and either the release of one or both of the products or a conformational change controls turnover [Grant, B., & Adams, J. A. (1996) Biochemistry 35, 2022-2029]. To determine which step or steps control turnover in the wild-type enzyme, we used a catalytic trapping technique to measure directly the dissociation rate constant for ADP. The phosphorylation of two peptide substrates, LRRASLG and GRTGRRNSI, was monitored using a rapid quench flow technique under conditions where saturating concentrations of ADP were preequilibrated with the enzyme before excess ATP and one of the substrates were added to trap the free enzyme and to start the phosphorylation reaction. Under ADP preequilibration conditions, no 'burst' phase was observed, and although the rate of linear, steady-state turnover was unaffected, the net production of phosphopeptide lagged behind the non-preequilibrated control. This phenomenon occurs due to the slow release of the product, and kinetic modeling suggests that this effect can be explained if the dissociation rate constant for ADP is 24 s-1 and solely limits turnover (kcat = 23 s-1) for the phosphorylation of LRRASLG. Using GRTGRRNSI, the dissociation rate constant for ADP is 35 s-1 and limits turnover (kcat = 29 s-1) if the reaction is initiated by the addition of enzyme. Under preequilibration conditions with either ATP or GRTGRRNSI, turnover is approximately 50% lower, suggesting that ADP release may partially control this parameter. This preequilibration effect can be explained by slowly interconverting enzyme forms with specific peptide-induced turnover properties. These studies indicate that ADP release is an essential rate-limiting component for turnover but also suggests that other factors contribute subtly when the structure of the substrate is altered.     

Deletion of a highly motional residue affects formation of the Michaelis complex for Escherichia coli dihydrofolate reductase

Analysis of the dihydrofolate reductase (DHFR) complex with folate by two-dimensional heteronuclear (1H-15N) nuclear magnetic relaxation revealed that isolated residues exhibit diverse backbone fluctuations on the nanosecond to picosecond time scale [Epstein, D. M., Benkovic, S. J., and Wright, P. E. (1995) Biochemistry 34, 11037-11048]. These dynamical features may be significant in forming the Michaelis complex. Of these residues, glycine 121 displays large-amplitude backbone motions on the nanosecond time scale. This amino acid, strictly conserved for prokaryotic DHFRs, is located at the center of the betaF-betaG loop. To investigate the catalytic importance of this residue, we report the effects of Gly121 deletion and glycine insertion into the modified betaF-betaG loop. Relative to wild type, deletion of Gly121 dramatically decreases the rate of hydride transfer 550-fold and the strength of cofactor binding 20-fold for NADPH and 7-fold for NADP+. Furthermore, DeltaG121 DHFR requires conformational changes dependent on the initial binary complex to attain the Michaelis complex poised for hydride transfer. Surprisingly, the insertion mutants displayed a significant decrease in both substrate and cofactor binding. The introduction of glycine into the modified betaF-betaG loop, however, generally eliminated conformational changes required by DeltaG121 DHFR to attain the Michaelis complex. Taken together, these results suggest that the catalytic role for the betaF-betaG loop includes formation of liganded complexes and proper orientation of substrate and cofactor. Through a transient interaction with the Met20 loop, alterations of the betaF-betaG loop can orchestrate proximal and distal effects on binding and catalysis that implicate a variety of enzyme conformations participating in the catalytic cycle.     

Isolation and characterization of thymitaq (AG337) and 5-fluoro-2-deoxyuridylate-resistant mutants of human thymidylate synthase from ethyl methanesulfonate-exposed human sarcoma HT1080 cells

Thymidylate synthase plays an essential role in the synthesis of DNA. Recently, several new and specific thymidylate synthase inhibitors that occupy the folate binding site, including Tomudex(R), BW1843U89, and Thymitaq, have demonstrated therapeutic activity in patients with advanced cancer. In order to find drug-resistant forms of human thymidylate synthase for gene therapy applications, human sarcoma HT1080 cells were exposed to ethyl methanesulfonate and Thymitaq selection. Thymitaq-resistant clonal derived sublines were established, and analysis indicated that both gene amplification and point mutations contributed to drug resistance. Eight mutant cDNAs that were identified from Thymitaq-resistant sublines were generated by site-directed mutagenesis and transfected into thymidylate synthase-negative cells. Only K47E, D49G, or G52S mutants retain enzyme activity. Moreover, cytotoxicity studies demonstrated that D49G and G52S transfected cells, besides displaying resistance to Thymitaq with IC50 values 40- and 12-fold greater than wild-type enzyme transfected cells, respectively, also lead to fluorodeoxyuridine resistance (26- and 97-fold in IC50 values, respectively) but not to Tomudex or BW1843U89. Characterization of the purified altered enzymes obtained from expression in Escherichia coli is consistent with the cell growth inhibition results. We postulate that the D49G or G52S mutation leads to the structural perturbation of the highly conserved Arg50 loop, decreasing the binding of thymidylate synthase to the inhibitors, Thymitaq and fluorodeoxyuridylate.     

Mutation of serine-46 to aspartate in the histidine-containing protein of Escherichia coli mimics the inactivation by phosphorylation of serine-46 in HPrs from gram-positive bacteria

Histidine-containing protein (HPr) is a phosphocarrier protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system. HPr is phosphorylated at the active site residue, His15, by phosphoenolpyruvate-dependent enzyme I in the first enzyme reaction in the process of phosphoryl transfer to sugar. In many Gram-positive bacterial species HPr may also be phosphorylated at Ser46 by an ATP-dependent protein kinase but not in the Gram-negative Escherichia coli and Salmonella typhimurium. One effect of the phosphorylation at Ser46 is to make HPr a poor acceptor for phosphorylation at His15. In Bacillus subtilis HPr, the mutation Ser46Asp mimics the effects of phosphorylation. A series of mutations were made at Ser46 in E. coli HPr: Ala, Arg, Asn, Asp, Glu, and Gly. The two acidic replacements mimic the effects of phosphorylation of Ser46 in HPrs from Gram-positive bacteria. In particular, when mutated to Asp46, the His 15 phosphoacceptor activity (enzyme I Km/Kcat) decreases by about 2000-fold (enzyme I Km, 4 mM HPr; Kcat, approximately 30%). The alanine and glycine mutations had near-wild-type properties, and the asparagine and arginine mutations yielded small changes to the Km values. The crystallographic tertiary structure of Ser46Asp HPr has been determined at 1.5 A resolution, and several changes have been observed which appear to be the effect of the mutation. There is a tightening of helix B, which is demonstrated by a consistent shortening of hydrogen bond lengths throughout the helix as compared to the wild-type structure. There is a repositioning of the Gly54 residue to adopt a 3(10) helical pattern which is not present in the wild-type HPr. In addition, the higher resolution of the mutant structure allows for a more definitive placement of the carbonyl of Pro11. The consequence of this change is that there is no torsion angle strain at residue 16. This result suggests that there is no active site torsion angle strain in wild-type E. coli HPr. The lack of substantial change at the active center of E. coli HPr Ser46Asp HPr suggests that the effect of the Ser46 phosphorylation in HPrs from Gram-positive bacteria is due to an electrostatic interference with enzyme I binding.     

Characterization of D10S and K71E mutants of human cytosolic hsp70

To determine the effect of mutations at the nucleotide-binding site of recombinant Hsp70 on its interaction with protein and peptide substrates, point mutations were made at D10 and K71, two residues at the active site. The D10S mutation weakened both ATP and ADP binding, while the K71E mutation weakened only ATP binding. In binding experiments using Hsp70 with no bound nucleotide, the mutated Hsp70s interacted with clathrin and peptide just like the wild-type Hsp70. However, the D10 mutation completely abolished the effects of both ATP and ADP on peptide and clathrin binding. The K71 mutation also abolished the effect of ATP on substrate binding, but ADP, which still bound tightly, had its normal effect on substrate binding. In addition, the D10S and K71E mutants had greatly reduced ability to uncoat clathrin-coated vesicles at pH 7.0, bind to clathrin baskets at pH 6.0, and undergo polymerization induced by YDJ1 in the presence of ATP. We conclude, first, that nucleotides must bind strongly to Hsp70 to affect substrate binding and, second, that interaction of Hsp70 with DnaJ homologues may also require a strongly bound ATP.     

Phosphorylation and functional properties of the IIA domain of the lactose transport protein of Streptococcus thermophilus

The lactose-H+ symport protein (LacS) of Streptococcus thermophilus has a carboxyl-terminal regulatory domain (IIALacS) that is homologous to a family of proteins and protein domains of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in various organisms, of which IIAGlc of Escherichia coli is the best-characterized member. On the basis of these similarities, it was anticipated that IIALacS would be able to perform one or more functions associated with IIAGlc, i.e., carry out phosphoryl transfer and/or affect other catabolic functions. The gene fragment encoding IIALacS was overexpressed in Escherichia coli, and the protein was purified in two steps by metal affinity and anion-exchange chromatography. IIALacS was unable to restore glucose uptake in a IIAGlc-deficient strain, which is consistent with a very low rate of phosphorylation of IIALacS by phosphorylated HPr (HPr approximately P) from E. coli. With HPr approximately P from S. thermophilus, the rate was more than 10-fold higher, but the rate constants for the phosphorylation of IIALacS (k1 = 4.3 x 10(2) M-1 s-1) and dephosphorylation of IIALacS approximately P by HPr (k-1 = 1.1 x 10(3) M-1 s-1) are still at least 4 orders of magnitude lower than for the phosphoryltransfer between IIAGlc and HPr from E. coli. This finding suggests that IIALacS has evolved into a protein domain whose main function is not to transfer phosphoryl groups rapidly. On the basis of sequence alignment of IIA proteins with and without putative phosphoryl transfer functions and the known structure of IIAGlc, we constructed a double mutant [IIALacS(I548E/G556D)] that was predicted to have increased phosphoryl transfer activity. Indeed, the phosphorylation rate of IIALacS(I548E/G556D) by HPr approximately P increased (k1 = 4.0 x 10(3) M-1 s-1) and became nearly independent of the source of HPr approximately P (S. thermophilus, Bacillus subtilis, or E. coli). The increased phosphoryl transfer rate of IIALacS(I548E/G556D) was insufficient to complement IIAGlc in PTS-mediated glucose transport in E. coli. Both IIALacS and IIALacS(I548E/G556D) could replace IIAGlc, but in another function: they inhibited glycerol kinase (inducer exclusion) when present in the unphosphorylated form.     

A mutant yeast topoisomerase II (top2G437S) with differential sensitivity to anticancer drugs in the presence and absence of ATP

To further characterize the mechanistic basis for cellular resistance/hypersensitivity to anticancer drugs, a yeast genetic system was used to select a mutant type II topoisomerase that conferred cellular resistance to CP-115,953, amsacrine, etoposide, and ellipticine. The mutant enzyme contained a single point mutation that converted Gly437 --> Ser (top2G437S). Purified top2G437S displayed wild-type enzymatic activity in the absence of drugs but exhibited two properties that were not predicted by the cellular resistance phenotype. First, in the absence of ATP, it was hypersensitive to all of the drugs examined and hypersensitivity correlated with increased drug affinity. Second, in the presence of ATP, top2G437S lost its hypersensitivity and displayed wild-type drug sensitivity. Since the resistance of yeast harboring top2G437S could not be explained by alterations in enzyme-drug interactions, physiological levels of topoisomerase II were determined. The Gly437 --> Ser mutation reduced the stability of topoisomerase II and decreased the cellular concentration of the enzyme. These findings suggest that the physiological drug resistance phenotype conferred by top2G437S results primarily from its decreased stability. This study highlights the need to analyze both the biochemistry and the physiology of topoisomerase II mutants with altered drug sensitivity in order to define the mechanistic bridge that links enzyme function to cellular phenotype.     

Genetic probing of the stalk segments associated with M2 and M3 of the plasma membrane H+-ATPase from Saccharomyces cerevisiae

The stalk region of the H+-ATPase from Saccharomyces cerevisiae has been proposed to play a role in coupling ATP hydrolysis to proton transport. Genetic probing was used to examine the role of stalk segments S2 and S3, associated with M2 and M3, respectively. Saturation mutagenesis was used to explore the role of side group character at position Ile183 in S2, at which an alanine substitution was shown previously to partially uncouple the enzyme (Wang, G., Tamas, M. J., Hall, M. J., Pascual-Ahuir, A., and Perlin, D. S. (1996) J. Biol. Chem. 271, 25438-25445). Diverse side group substitutions were tolerated at this position, although three substitutions, I183N, I183R, and I183Y required second site mutations at the C terminus of the enzyme for stabilization. Substitution of glycine and proline at Ile183 resulted in lethal phenotypes, suggesting that the backbone may be more important than side group at this position. Proline/glycine mutagenesis was used to study additional sites in S2 and S3. The substitution of proline at Gly186 resulted in a lethal phenotype, whereas substitutions in S3 of proline or serine at Gly270 and proline or glycine at Thr287 resulted in viable mutants. Mutations G270P and T287P resulted in mutant enzymes that produced pronounced growth defects and ATP hydrolysis rates that were 35% and 60% lower than wild type enzyme, respectively. The mutant enzymes transported protons at rates consistent with their ATPase activity, suggesting that the growth defects observed were due to a reduced rate of ATP hydrolysis and not to uncoupling of proton transport. The prominent growth phenotypes produced by mutations G270P and T287P permitted the isolation of suppressor mutations, which restored wild type growth. Most of the suppressors either replaced the primary site mutation with alanine or restored the wild type residue by ectopic recombination with PMA2, both of which restore alpha-helical tendency. This study suggests that maintaining alpha-helical character is essential to S2 and may play an important role in S3.     

A recombinant bisphosphoglycerate mutase variant with acid phosphatase homology degrades 2,3-diphosphoglycerate

To date no definite and undisputed treatment has been found for sickle cell anemia, which is characterized by polymerization of a deoxygenated hemoglobin mutant (HbS) giving rise to deformed erythrocytes and vasoocclusive complications. Since the erythrocyte glycerate 2,3-bisphosphate (2,3-DPG) has been shown to facilitate this polymerization, one therapeutic approach would be to decrease the intraerythrocytic level of 2,3-DPG by increasing the phosphatase activity of the bisphosphoglycerate mutase (BPGM; 3-phospho-D-glycerate 1,2-phosphomutase, EC 5.4.2.4). For this purpose, we have investigated the role of Gly-13, which is located in the active site sequence Arg9-His10-Gly11-Glu12-Gly13 in human BPGM. This sequence is similar to the Arg-His-Gly-Xaa-Arg* sequence of the distantly related acid phosphatases, which catalyze as BPGM similar phosphoryl transfers but to a greater extent. We hypothesized that the conserved Arg* residue in acid phosphatase sequences facilitates the phosphoryl transfer. Consequently, in human BPGM, we replaced by site-directed mutagenesis the corresponding amino acid residue Gly13 with an Arg or a Lys. In another experiment, we replaced Gly13 with Ser, the amino acid present at the corresponding position of the homologous yeast phosphoglycerate mutase (D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1). Mutation of Gly13 to Ser did not modify the synthase activity, whereas the mutase and the phosphatase were 2-fold increased or decreased, respectively. However, replacing Gly13 with Arg enhanced phosphatase activity 28.6-fold, whereas synthase and mutase activities were 10-fold decreased. The presence of a Lys in position 13 gave rise to a smaller increase in phosphatase activity (6.5-fold) but an identical decrease in synthase and mutase activities. Taken together these results support the hypothesis that a positively charged amino acid residue in position 13, especially Arg, greatly activates the phosphoryl transfer to water. These results also provide elements for locating the conserved Arg* residue in the active site of acid phosphatases and facilitating the phosphoryl transfer. The implications for genetic therapy of sickle cell disease are discussed.     

Kinetic mechanism of monomeric non-claret disjunctional protein (Ncd) ATPase

The non-claret disjunctional protein (Ncd) is a kinesin-related microtubule motor that moves toward the negative end of microtubules. The kinetic mechanism of the monomer motor domain, residues 335-700, satisfied a simple scheme for the binding of 2'-3'-O-(N-methylanthraniloyl) (MANT) ATP, the hydrolysis step, and the binding and release of MANT ADP, where T, D, and Pi refer to nucleotide triphosphate, nucleotide diphosphate, and inorganic phosphate, respectively, and MtN is the complex of an Ncd motor domain with a microtubule site. Rate constants k1 and k-4 are the rates of a first order step, an isomerization induced by nucleotide binding. The apparent second order rate constants for the binding steps are 1.5 x 10(6) M-1 s-1 for MANT ATP and 3.5 x 10(6) M-1 s-1 for MANT ADP (conditions, 50 mM NaCl, pH 6.9, 21 degrees C). The rate constant of the hydrolysis step (k2) was obtained from quench flow measurements of the phosphate burst phase corrected for the contribution of the rate of product release to the transient rate constant. The rate of phosphate dissociation was not measured; the value was assigned to account for a steady state rate of 3 s-1. The MtN complex is dissociated by ATP at a rate of 10 s-1 based on light scattering measurements. Dissociation constants of Ncd-nucleotide complexes from microtubules increased in the order adenosine 5'-O-(thiotriphosphate) (ATPgammaS) < ADP-AlF4 < ATP < ADP < ADP-vanadate. Comparison of the properties of Ncd with a monomeric kinesin K332 (Ma and Taylor (1997) J. Biol. Chem. 272, 717-723) showed a close similarity, except that the rate constants for the hydrolysis and ADP release steps and the steady state rate are approximately 15-20 times smaller for Ncd. There are two differences that may affect the reaction pathway. The rate of dissociation of MtN by ATP is comparable to the rate of the hydrolysis step, and N.T may dissociate in the cycle, whereas for kinesin, dissociation occurs after hydrolysis. The rate of dissociation of MtN by ADP is larger than the rate of ADP release from MtN.D, whereas for the microtubule-kinesin complex, the rate of dissociation by ADP is smaller than the rate of ADP release. The monomeric Mt.Ncd complex is not processive.     

Identification of 19 protein S gene mutations in patients with phenotypic protein S deficiency and thrombosis. Protein S Study Group

Protein S is a protein C-dependent and independent inhibitor of the coagulation cascade. Deficiency of protein S is an established risk factor for venous thromboembolism. We have used a strategy of specific amplification of the coding regions and intron/exon boundaries of the active protein S gene (PROS1) and direct single-strand solid phase sequencing, to seek mutations in 35 individuals with phenotypic protein S deficiency. Nineteen point mutations (16 novel) in 19 probands (or relatives of probands) with venous thromboembolism are reported here. Fifteen of the 19 mutations were expected to be causal and included 10 missense mutations (Lys9Glu, Glu26Ala, Gly54Glu, Cys145Tyr, Cys200Ser, Ser283Pro, Gly340Asp, Cys408Ser, Ser460Pro, and Cys625Arg). Three of the 15 mutations resulted in premature stop codons (delete T 635 producing a stop codon at position 126, Lys368stop and Tyr595stop) and two were at intron/exon boundaries (+1 G to A in intron d and +3 A to C in intron j). Of the remaining four mutations, three were within intronic sequence and one was a silent mutation within the coding region and did not alter amino acid composition. In two of the 10 missense mutations, reduced plasma protein S activity compared with antigen level suggested the presence of variant (type II) protein S.     

His1069Gln and six novel Wilson disease mutations: analysis of relevance for early diagnosis and phenotype

In the present study we examined 33 German and 10 Cuban unrelated Wilson disease (WND) index patients and their relatives. The common His1069Gln mutation accounted for 42% of all WND chromosomes in the German series and the haplotype C was found to be highly predictive for this mutation. Six WND gene mutations have not been described previously and involved a splice site at intron 18 (3903 + del1G), a termination codon in the copper-binding region of exon 2 (Cys271X), and missense mutations in transmembrane region 2 (Gly710Ala), in transmembrane region 3 (Tyr741Cys), in the DKTGT motif (Thr1031Ile) and in the ATP loop region (Gly1176Arg). In 15 German WND index patients and three sibs both WND mutations could be determined and a genotype-phenotype correlation was attempted. Patients homozygous for the His1069Gln mutation showed almost the complete range of clinical presentations, and thus in our study this mutation is not associated with a late, neurological presentation.     

High resolution crystal structure of a human tumor necrosis factor-alpha mutant with low systemic toxicity

A human tumor necrosis factor-alpha (TNF-alpha) mutant (M3S) with low systemic toxicity in vivo was designed, and its structures in two different crystal packings were determined crystallographically at 1.8 and 2.15-A resolution, respectively, to explain altered biological activities of the mutant. M3S contains four changes: a hydrophilic substitution of L29S, two hydrophobic substitutions of S52I and Y56F, and a deletion of the N-terminal seven amino acids that is disordered in the structure of wild-type TNF-alpha. Compared with wild-type TNF-alpha, it exhibits 11- and 71-fold lower binding affinities for the human TNF-R55 and TNF-R75 receptors, respectively, and in vitro cytotoxic effect and in vivo systemic toxicity of M3S are 20 and 10 times lower, respectively. However, in a transplanted solid tumor mouse model, M3S suppresses tumor growth more efficiently than wild-type TNF-alpha. M3S is highly resistant to proteolysis by trypsin, and it exhibits increased thermal stability and a prolonged half-life in vivo. The L29S mutation causes substantial restructuring of the loop containing residues 29-36 into a rigid segment as a consequence of induced formation of intra- and intersubunit interactions, explaining the altered receptor binding affinity and thermal stability. A mass spectrometric analysis identified major proteolytic cleavage sites located on this loop, and thus the increased resistance of M3S to the proteolysis is consistent with the increased rigidity of the loop. The S52I and Y56F mutations do not induce a noticeable conformational change. The side chain of Phe56 projects into a hydrophobic cavity, while Ile52 is exposed to the bulk solvent. Ile52 should be involved in hydrophobic interactions with the receptors, since a mutant containing the same mutations as in M3S except for the L29S mutation exhibits an increased receptor binding affinity. The low systemic toxicity of M3S appears to be the effect of the reduced and selective binding affinities for the TNF receptors, and the superior tumor-suppression of M3S appears to be the effect of its weak but longer antitumoral activity in vivo compared with wild-type TNF-alpha. It is also expected that the 1.8-A resolution structure will serve as an accurate model for explaining the structure-function relationship of wild-type TNF-alpha and many TNF-alpha mutants reported previously and for the design of new TNF-alpha mutants.     

Mutagenesis of some positive and negative residues occurring in repeat triad residues in the ADP/ATP carrier from yeast

In AAC2 from Saccharomyces cerevisiae, nine additional charged residues (six positive, three negative) were neutralized by mutagenesis following the previous mutation of six arginines. Oxidative phosphorylation (OxPhos) in cells and mitochondria, the expression level of AAC protein, and the various transport modes of AAC in the reconstituted system were measured. Mutations are: within the first helix at K38A which is exclusive for AAC; K48A, and R152A, part of a positive triad occurring in the matrix portion of each repeat; two matrix lysines, K179M and K182I, and the negative triad helix-terminating residues, E45G, D149S, D249S. Cellular ATP synthesis (OxPhos) is nearly completely inhibited in K48A, R152A, D149S, and D249S, but still amounts to 10% in K38A and between 30% and 90% in the gly+ mutants K179M, K179I + K182I, and E45G. Comparison of the AAC content measured by ELISA and the binding of [3H]CAT and [3H]BKA reveals discrepancies in K48A, D149S, and D249S mitochondria, which provide evidence that these mutations largely abolish inhibitor binding. Also these mitochondria have undetectable OxPhos. Differently in K38A, CAT and BKA binding are retained at high AAC levels but OxPhos is very low. This reveals a special functional role of K38, different from the more structural role of R152, K48, D149, and D249. Transport activity was measured with reconstituted AAC. The electroneutral ADP/ADP exchange of gly- mutants is largely or fully suppressed in K48A, D149S, and D249S. K38A and R152A are still active at 18% and 30% of wt. The other three exchange modes, ATP/ADP, ADP/ATP, and ATP/ATP, are nearly suppressed in all gly- mutants but remain high in gly+ mutants. ATP-linked modes are higher than the ADP/ADP mode in gly+ but lower in gly- mutants, resulting in an exchange mode inversion (EMI). In the competition for AAC2 transport capacity, the weak ATP exporting modes are suppressed by the much stronger unproductive ADP/ADP mode causing inhibition of OxPhos. Together with previous results all members of three charge triads are now mutagenized, revealing drastic functional rotatory asymmetries within the three repeat domains. In the intrahelical arginine triad the third (R294A), in the positive matrix triad the second (R152A), and in the helix-terminating negative triad the first (E45G) still show high activity.     

Interaction of mant-adenosine nucleotides and magnesium with kinesin

Displacement of the fluorescent substrate analogue methylanthraniloyl ADP (mant-ADP) from kinesin by excess ATP results in a biphasic fluorescent transient. The pH and microtubule dependence of the rates and amplitudes indicates that the two phases are produced by release of bound mant-ADP, with an excess of the 3'-isomer, followed by the subsequent relaxation of the free 2'- and 3'-isomers to their equilibrium distribution. The first phase for release of mant-ADP is accelerated by microtubules and occurs at the same rate as ADP release measured using [32P]ADP. The second phase is subject to base catalysis and occurs at the same rate as the isomerization of isolated 2'- or 3'-mant-ATP over a 100-fold range of rates. The bound mant-ADP isomers undergo isomerization rapidly when bound to kinesin at pH 8.2, whereas mant-ADP isomers interconvert only slowly when bound to myosin. No fluorescence resonance energy transfer occurs between the single tryptophan in the kinesin neck domain and bound mant-ADP, but efficient energy transfer does occur from protein tyrosine groups. The rate of mant-ADP release in the absence of microtubules is minimal (0.005 s-1) at pH 7-8, 2 mM Mg2+, and 25 mM KCl but is accelerated at lower pH (0.04 s-1 at pH 5.5) and either lower or higher [KCl] (0.01 and 0. 06 s-1 at 0 and 800 mM KCl, respectively). The microtubule-stimulated rate of ADP release is accelerated at low pH and inhibited by high concentrations of monovalent salts. Reduction of the free Mg2+ by addition of excess EDTA increases the release of mant-ADP from E.MgADP to 0.03 s-1. This acceleration at low Mg2+ likely represents sequential release of Mg2+ at 0.03 s-1 followed by rapid release of ADP, as the rate of ADP release from Mg-free E.ADP is fast (>0.5 s-1). At high Mg2+, rebinding of Mg2+ to E.ADP forces release of ADP from the E.MgADP complex at 0.005 s-1.     

Interaction of F-actin with synthetic peptides spanning the loop region of human cardiac beta-myosin heavy chain containing Arg403

The atomic model of the F-actin-myosin subfragment 1 complex (acto-S-1) from skeletal muscle suggests that the transition of the complex from a weakly to a strongly binding state, generating mechanical force during the contractile cycle, may involve the attachment of the upper 50-kDa subdomain of myosin subfragment 1 (S-1) to the interface between subdomains 1 and 3 of actin. For the human cardiac myosin, this putative interaction would take place at the ordered loop including Arg403 of the beta-heavy chain sequence, a residue whose mutation into Gln is known to elicit a severe hypertrophic cardiomyopathy caused by a decrease of the rate of the actomyosin ATPase activity. Moreover, in several nonmuscle myosins the replacement of a Glu residue within the homolog loop by Ser or Thr also results in the reduction of the actomyosin ATPase rate that is alleviated by phosphorylation. As an approach to the characterization of the unknown interaction properties of F-actin with this particular S-1 loop region, we have synthesized four 17-residue peptides corresponding to the sequence Gly398-Gly414 of the human beta-cardiac myosin. Three peptides included Arg403 (GG17) or Gln403 (GG17Q) or Ser409 (GG17S) and the fourth peptide (GG17sc) was a scrambled version of the normal GG17 sequence. Using fluorescence polarization, cosedimentation analyses and photocross-linking, we show that the three former peptides, but not the scrambled sequence, directly associate in solution to F-actin, at a nearly physiological ionic strength, with almost identical affinities (Kd approximately 40 microM). The binding strength of the F-actin-GG17 peptide complex was increased fivefold (Kd = 8 microM) in the presence of subsaturating concentrations of added skeletal S-1 relative to actin, without apparent competition between the peptide and S-1. Each of the three actin-binding peptides inhibited the steady-state actin-activated MgATPase of skeletal S-1 by specifically decreasing about twofold the Vmax of the reaction without changing the actin affinity for the S-1-ATP intermediate. Cosedimentation assays indicated the binding of about 0.65 mol peptide/mol actin under conditions inducing 70% inhibition. Collectively, the data point to a specific and stoichiometric interaction of the peptides with F-actin that uncouples its binding to S-1 from ATP hydrolysis, probably by interfering with the proper attachment of the S-1 loop segment to the interdomain connection of actin.     

Infectious disease and sociodemographic characteristics of foreign immigrants in the Central Penitentiary for Men in Barcelona

A number of thrombin mutants have been constructed to investigate the role of Trp96 and the beta-insertion loop for the specificity of thrombin. Thrombin(60D) consists of the replacement of the beta-insertion loop (14 amino acid residues from 59 to 63, including a 9-residue insertion at position 60) with the corresponding four residues in trypsin, Tyr-Lys-Ser-Gly; thrombin(GGG) is a smaller loop mutation in which the residues Tyr(60A)Pro(60B)Pro(60C)Trp(60D) Asp(60E)Lys(60F) of the beta-insertion loop were replaced by Gly-Gly-Gly; thrombin(96S) consists of a point mutation Trp96 --> Ser; and thrombin(GGG/96S) is the double mutant incorporating both changes. Thrombin(96S) clots fibrinogen approximately 3 times more slowly than thrombin, with the two beta-insertion loop mutants, thrombin(GGG) and thrombin(GGG/96S), reacting approximately 3000- and 1300-fold more slowly, respectively. The specificity constant kcat/Km for the cleavage of fibrinopeptide A and fibrinopeptide B by thrombin(96S) was 2.6 and 0.35 microM(-1) s(-1), respectively, compared to 10 and 2.5 microM(-1) s(-1) for wild-type recombinant thrombin, respectively. Kinetic constants were determined for the hydrolysis of H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline. The Michaelis constant Km increased approximately 6-fold for thrombin(96S) and >200-fold for thrombin(GGG) and thrombin(GGG/96S) when compared to wild-type recombinant thrombin, while the catalytic constant kcat remained approximately the same. All mutants were more susceptible to inhibition by BPTI than wild-type recombinant thrombin. Clearly, the beta-insertion loop is important for thrombin activity. But the mutation of Trp96 --> Ser can compensate somewhat for the loss of binding at the beta-insertion loop. The deletion of the hydrophobic interaction between Trp96 and Pro(60B)Pro(60C) appears to decrease the stability of the beta-insertion loop, thereby causing a decrease in binding efficiency.     

The Escherichia coli FOF1 gammaM23K uncoupling mutant has a higher K0.5 for Pi. Transition state analysis of this mutant and others reveals that synthesis and hydrolysis utilize the same kinetic pathway

The Escherichia coli FOF1 ATP synthase uncoupling mutation, gammaM23K, was found to increase the energy of interaction between gamma and beta subunits, prevent the proper utilization of binding energy to drive catalysis, and block the enzyme in a Pi release mode. In this paper, the effects of this mutation on substrate binding in cooperative ATP synthesis are assessed. Activation of ATP synthesis by ADP and Pi was determined for the gammaM23K FOF1. The K0.5 for ADP was not affected, but K0.5 for Pi was approximately 7-fold higher even though the apparent Vmax was close to the wild-type level. Wild-type enzyme had a turnover number of 82 s-1 at pH 7.5 and 30 degrees C. During oxidative phosphorylation, the apparent dissociation constant (KI) for ATP was not affected and was 5-6 mM for both wild-type and gammaM23K enzymes. Thus, the apparent binding affinity for ATP in the presence of DeltamuH+ was lowered by 7 orders of magnitude from the affinity measured at the high-affinity catalytic site. Arrhenius analysis of ATP synthesis for the gammaM23K FOF1 revealed that, like those of ATP hydrolysis, the transition state DeltaH was much more positive and TDeltaS was much less negative, adding up to little change in DeltaG. These results suggested that ATP synthesis is inefficient because of an extra bond between gamma and beta subunits which must be broken to achieve the transition state. Analysis of the transition state structures using isokinetic plots demonstrate that ATP hydrolysis and synthesis utilize the same kinetic pathway. Incorporating this information into a model for rotational catalysis suggests that at saturating substrate concentrations, the rate-limiting step for hydrolysis and synthesis is the rotational power stroke where each of the beta subunits changes conformation and affinity for nucleotide.     

Amino acid sequence environment modulates the disruption by osteogenesis imperfecta glycine substitutions in collagen-like peptides

Ostoegenesis imperfecta (OI) or "brittle bone" disease is associated with mutations in the genes for type I collagen chains and produces variable phenotypes, ranging from lethal cases at birth to mild cases with increased bone fractures. The most common OI mutations are single base substitutions leading to replacement of Gly by another residue, breaking the typical (Gly-X-Y)n repeating sequence pattern of the collagen triple-helix. Triple-helical peptides were designed to focus on residues 892-921 of the alpha1 chain of type I collagen, where two OI Gly-->Ser mutations are found in close proximity, a mild mutation at site 901 and a lethal mutation at site 913. Peptides were designed to include amino acid sequences around these mutation sites, and were synthesized with the normal sequence or with the Gly-->Ser mutated sequence. The peptide including the normal sequence residues 892-909 with four Gly-Pro-Hyp triplets at the C-terminus formed a stable triple-helix, and introduction of a Ser residue for Gly at the 901 mutation site led to a 50% loss of triple-helix content and a decrease in thermal stability, with little effect on folding. A peptide including residues 904-921 again formed a stable triple-helix, but the introduction of the Gly-->Ser substitution at site 913 led to a much greater decrease in thermal stability. These studies demonstrate the impact of local sequences flanking the Gly substitution on structural consequences and support the concept of variability and regional effects along the collagen molecule.     

Decoupling of the bc1 complex in S. cerevisiae; point mutations affecting the cytochrome b gene bring new information about the structural aspect of the proton translocation

Four mutations in the mitochondrial cytochrome b of S. cerevisiae have been characterized with respect to growth capacities, catalytic properties, ATP/2e- ratio, and transmembrane potential. The respiratory-deficient mutant G137E and the three pseudo-wild type revertants E137 + I147F, E137 + C133S, and E137 + N256K were described previously (Tron and Lemesle-Meunier, 1990; Di Rago et al., 1990a). The mutant G137E is unable to grow on respiratory substrates but its electron transfer activity is partly conserved and totally inhibited by antimycin A. The secondary mutations restore the respiratory growth at variable degree, with a phosphorylation efficiency of 12-42% as regards the parental wild type strain, and result in a slight increase in the various electron transfer activities at the level of the whole respiratory chain. The catalytic efficiency for ubiquinol was slightly (G137E) or not affected (E137 + I147F, E137 + C133S, and E137 + N256K) in these mutants. Mutation G137E induces a decrease in the ATP/2e- ratio (50% of the W.T. value) and transmembrane potential (60% of the W.T. value) at the bc1 level, whereas the energetic capacity of the cytochrome oxidase is conserved. Secondary mutations I147F, C133S, and N256K partly restore the ATP/2e- ratio and the transmembrane potential at the bc1 complex level. The results suggest that a partial decoupling of the bc1 complex is induced by the cytochrome b point mutation G137E. In the framework of the protonmotive Q cycle, this decoupling can be explained by the existence of a proton wire connecting centers P and N in the wild type bc1 complex which may be amplified or uncovered by the G137E mutation when the bc1 complex is functioning.