Inhaled anesthetic binding sites in human serum albumin

Previous evidence suggests multiple anesthetic binding sites on human serum albumin, but to date, we have only identified Trp-214 in an interdomain cleft as contributing to a binding site. We used a combination of site-directed mutagenesis, photoaffinity labeling, amide hydrogen exchange, and tryptophan fluorescence spectroscopy to evaluate the importance to binding of a large domain III cavity and compare it to binding character of the 214 interdomain cleft. The data show anesthetic binding in this domain III cavity of similar character to the interdomain cleft, but selectivity for different classes of anesthetics exists. Occupancy of these sites stabilizes the native conformation of human serum albumin. The features necessary for binding in the cleft appear to be fairly degenerate, but in addition to hydrophobicity, there is evidence for the importance of polarity. Finally, myristate isosterically competes with anesthetic binding in the domain III cavity and allosterically enhances anesthetic binding in the interdomain cleft.     

The HPr kinase from Bacillus subtilis is a homo-oligomeric enzyme which exhibits strong positive cooperativity for nucleotide and fructose 1,6-bisphosphate binding

Carbon catabolite repression allows bacteria to rapidly alter the expression of catabolic genes in response to the availability of metabolizable carbon sources. In Bacillus subtilis, this phenomenon is controlled by the HPr kinase (HprK) that catalyzes ATP-dependent phosphorylation of either HPr (histidine containing protein) or Crh (catabolite repression HPr) on residue Ser-46. We report here that B. subtilis HprK forms homo-oligomers constituted most likely of eight subunits. Related to this complex structure, the enzyme displays strong positive cooperativity for the binding of its allosteric activator, fructose 1,6-bisphosphate, as evidenced by either kinetics of its phosphorylation activity or the intrinsic fluorescence properties of its unique tryptophan residue, Trp-235. It is further shown that activation of HPr phosphorylation by fructose 1,6-bisphosphate essentially occurs at low ATP and enzyme concentrations. A positive cooperativity was also detected for the binding of natural nucleotides or their 2'(3')-N-methylanthraniloyl derivatives, in either phosphorylation or fluorescence experiments. Most interestingly, quenching of the HprK tryptophan fluorescence by using either iodide or acrylamide revealed a heterogeneity of tryptophan residues within the population of oligomers, suggesting that the enzyme exists in two different conformations. This result suggests a concerted-symmetry model for the catalytic mechanism of positive cooperativity displayed by HprK.     

Roles of the exposed aromatic residues in crystalline chitin hydrolysis by chitinase A from Serratia marcescens 2170

Four exposed aromatic residues, two in the N-terminal domain (Trp-69 and Trp-33) and two in the catalytic domain (Trp-245 and Phe-232) of Serratia marcescens chitinase A, are linearly aligned with the deep catalytic cleft. To investigate the importance of these residues in the binding activity and hydrolyzing activity against insoluble chitin, site-directed mutagenesis to alanine was carried out. The substitution of Trp-69, Trp-33, or Trp-245 significantly reduced the binding activity to both highly crystalline beta-chitin and colloidal chitin. The substitution of Phe-232, which is located closest to the catalytic cleft, did not affect the binding activity. On the other hand, the hydrolyzing activity against beta-chitin microfibrils was significantly reduced by the substitution of any one of the four aromatic residues including Phe-232. None of the mutations reduced the hydrolyzing activity against soluble substrates. These results clearly demonstrate that the four exposed aromatic residues are essential determinants for crystalline chitin hydrolysis. Three of them, two in the N-terminal domain and one in the catalytic domain, play vital roles in the chitin binding. Phe-232 appeared to be important for guiding the chitin chain into the catalytic cleft. Based on these observations, a model for processive hydrolysis of crystalline chitin by chitinase A is proposed.     

Fluorescence of tryptophan residues in firefly luciferases and enzyme--substrate complexes

Quenching of tryptophan fluorescence of Luciola mingrelica (single tryptophan residue, Trp-419) and Photinus pyralis (two tryptophan residues, Trp-417 and Trp-426) luciferases with different quenchers (I-, Cs+, acrylamide) was studied. The conserved Trp-417(419) residue was shown to be not accessible to charged particles, and positively and negatively charged amino acid residues are located in close vicinity to it. We found previously unreported effective energy transfer from this tryptophan to luciferin during the quenching of the tryptophan fluorescence. The distance between the luciferin molecule and Trp-417(419) was calculated: 11-15 and 12-17 A for P. pyralis and L. mingrelica luciferases, respectively. The role of the conserved Trp residue in the catalysis is discussed. ATP and AMP are also quenchers of the tryptophan fluorescence of the luciferases. In this case, an allosteric mechanism of the interaction of Trp-417(419) with an excess of ATP (AMP) is proposed.     

A fluorescence investigation of the active site of Pseudomonas aeruginosa exotoxin A.

Single tryptophan mutant proteins of a catalytically active domain III recombinant protein (PE24) from Pseudomonas aeruginosa exotoxin A were prepared by site-directed mutagenesis. The binding of the dinucleotide substrate, NAD+, to the PE24 active site was studied by exploiting intrinsic tryptophan fluorescence for the wild-type, single Trp, and tryptophan-deficient mutant proteins. Various approaches were used to study the substrate binding process, including dynamic quenching, CD spectroscopy, steady-state fluorescence emission analysis, NAD+-glycohydrolase activity, NAD+ binding analysis, protein denaturation experiments, fluorescence lifetime analysis, steady-state anisotropy measurement, stopped flow fluorescence spectroscopy, and quantum yield determination. It was found that the conservative replacement of tryptophan residues with phenylalanine had little or no effect on the folded stability and enzyme activity of the PE24 protein. Dynamic quenching experiments indicated that when bound to the active site of the enzyme, the NAD+ substrate protected Trp-558 from solvent to a large extent but had no effect on the degree of solvent exposure for tryptophans 417 and 466. Also, upon substrate binding, the anisotropy of the Trp-417(W466F/W558F) protein showed the largest increase, followed by Trp-466(W417F/W558F), and there was no effect on Trp-558(W417F/W466F). Furthermore, the intrinsic tryptophan fluorescence exhibited the highest degree of substrate-induced quenching for the wild-type protein, followed in decreasing order by Trp-417(W466F/W558F), Trp-558(W417F/W466F), and Trp-466(W417F/W558F). These data provide evidence for a structural rearrangement in the enzyme domain near Trp-417 invoked by the binding of the NAD+ substrate.     

Functional and fluorescence analyses of tryptophan residues in H+-pyrophosphatase of Clostridium tetani

Homodimeric proton-translocating pyrophosphatase (H⁺-PPase; EC 3.6.1.1) maintains the cytoplasmic pH homeostasis of many bacteria and higher plants by coupling pyrophosphate (PP_i) hydrolysis and proton translocation. H⁺-PPase accommodates several essential motifs involved in the catalytic mechanism, including the PP_i binding motif and Acidic I and II motifs. In this study, 3 intrinsic tryptophan residues, Trp-75, Trp-365, and Trp-602, in H⁺-PPase from Clostridium tetani were used as internal probes to monitor the local conformational state of the periplasm domain, transmembrane region, and cytoplasmic domain, respectively. Upon binding of the substrate analog Mg-imidodiphosphate (Mg-IDP), local structural changes prevented the modification of tryptophan residues by N-bromosuccinimide (NBS), especially at Trp-602. Following Mg-P_i binding, Trp-75 and Trp-365, but not Trp-602, were slightly protected from structural modifications by NBS. These results reveal the conformation of H⁺-PPase is distinct in the presence of different ligands. Moreover, analyses of the Stern-Volmer relationship and steady-state fluorescence anisotropy also indicate that the local structure around Trp-602 is more exposed to solvent and varied under different environments. In addition, Trp-602 was identified to be a crucial residue in the H⁺-PPase that may potentially be involved in stabilizing the structure of the catalytic region by site-directed mutagenesis analysis.     

Frequency domain fluorescence studies of yeast phosphoglycerate kinase and its ternary complex

A frequency domain fluorescence study of yeast phosphoglycerate kinase has been performed to observe the effect of substrates on the structure and dynamics of the enzyme. At 20 degrees C and pH 7.2, a biexponential decay is observed for tryptophanyl emission. The short fluorescence lifetime (0.4 ns) component is associated with a spectrum having a 329-nm maximum and a 18.4-kJ/mol activation energy, Ea, for thermal quenching. The long-lifetime (3.5 ns) component has a 338-nm maximum and an Ea of only 7.9 kJ/mol. Tentatively we assign the short and long-lifetime components to Trp-333 and Trp-308. Binding of the substrates ATP and 3-phosphoglycerate leads to a significant increase in the fluorescence lifetime, the red shift of the emission spectrum and in the decrease in the Ea for both components. Acrylamide-quenching studies indicate that the two tryptophan residues have about the same degree of kinetic exposure to the quencher and that the binding of the substrates causes a very slight change in the quenching pattern. These fluorescence studies indicate that the binding of the substrates to phosphoglycerate kinase may influence the conformational dynamics around the two tryptophan residues located on one of the protein's domains.     

Actin-induced closure of the actin-binding cleft of smooth muscle myosin

The putative actin-binding interface of myosin is separated by a large cleft that extends into the base of the nucleotide binding pocket, suggesting that it may be important for mediating the nucleotide-dependent changes in the affinity for myosin on actin. We have genetically engineered a truncated version of smooth muscle myosin containing the motor domain and the essential light chain-binding region (MDE), with a single tryptophan residue at position 425 (F425W-MDE) in the actin-binding cleft. Steady-state fluorescence of F425W-MDE demonstrates that Trp-425 is in a more solvent-exposed conformation in the presence of MgATP than in the presence of MgADP or absence of nucleotide, consistent with closure of the actin-binding cleft in the strongly bound states of MgATPase cycle for myosin. Transient kinetic experiments demonstrate a direct correlation between the rates of strong actin binding and the conformation of Trp-425 in the actin-binding cleft, and suggest the existence of a novel conformation of myosin not previously seen in solution or by x-ray crystallography. Thus, these results directly demonstrate that: 1) the conformation of the actin-binding cleft mediates the affinity of myosin for actin in a nucleotide-dependent manner, and 2) actin induces conformational changes in myosin required to generate force and motion during muscle contraction.     

The roles of tryptophans in primer synthesis by the DNA primase of bacteriophage T7

DNA primases catalyze the synthesis of oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Prokaryotic primases consist of a zinc-binding domain (ZBD) necessary for recognition of a specific template sequence and a catalytic RNA polymerase domain. Interactions of both domains with the DNA template and ribonucleotides are required for primer synthesis. Five tryptophan residues are dispersed in the primase of bacteriophage T7: Trp-42 in the ZBD and Trp-69, -97, -147, and -255 in the RNA polymerase domain. Previous studies showed that replacement of Trp-42 with alanine in the ZBD decreases primer synthesis, whereas substitution of non-aromatic residues for Trp-69 impairs both primer synthesis and delivery. However, the roles of tryptophan at position 97, 147, or 255 remain elusive. To investigate the essential roles of these residues, we replaced each tryptophan with the structurally similar tyrosine and examined the effect of this subtle alteration on primer synthesis. The substitution at position 42, 97, or 147 reduced primer synthesis, whereas substitution at position 69 or 255 did not. The functions of the tryptophans were further examined at each step of primer synthesis. Alteration of residue 42 disturbed the conformation of the ZBD and resulted in partial loss of the zinc ion, impairing binding to the ssDNA template. Replacement of Trp-97 with tyrosine reduced the binding affinity to NTP and the catalysis step. The replacement of Trp-147 with tyrosine also impaired the catalytic step. Therefore, Trp-42 is important in maintaining the conformation of the ZBD for template binding; Trp-97 contributes to NTP binding and the catalysis step; and Trp-147 maintains the catalysis step.     

Regulation of the catalytic function of coagulation factor VIIa by a conformational linkage of surface residue Glu 154 to the active site

Coagulation factor VIIa is an allosterically regulated trypsin-like serine protease that initiates the coagulation pathways upon complex formation with its cellular receptor and cofactor tissue factor (TF). The analysis of a conformation-sensitive monoclonal antibody directed to the macromolecular substrate exosite in the VIIa protease domain demonstrated a conformational link from this exosite to the catalytic cleft that is independent of cofactor-induced allosteric changes. In this study, we identify Glu 154 as a critical surface-exposed exosite residue side chain that undergoes conformational changes upon active site inhibitor binding. The Glu 154 side chain is important for hydrolysis of scissile bond mimicking peptidyl p-nitroanilide substrates, and for inhibition of VIIa's amidolytic function upon antibody binding. This exosite residue is not linked to the catalytic cleft residue Lys 192 which plays an important role in thrombin's allosteric coupling to exosite I. Allosteric linkages between VIIa's active site and the cofactor binding site or between the cofactor binding site and the macromolecular substrate exosite were not influenced by mutation of Glu 154. Glu 154 thus only influences the linkage of the macromolecular substrate binding exosite to the catalytic center. These data provide novel evidence that allosteric regulation of VIIa's catalytic function involves discrete and independent conformational linkages and that allosteric transitions in the VIIa protease domain are not globally coupled.     

Fluorescence analysis of calmodulin mutants containing tryptophan: conformational changes induced by calmodulin-binding peptides from myosin light chain kinase and protein kinase II.

Peptide-induced conformational changes in five isofunctional mutants of calmodulin (CaM), each bearing a single tryptophan residue either at the seventh position of each of the four calcium-binding loops (i.e., amino acids 26, 62, 99, and 135) or in the central helix (amino acid 81) were studied by using fluorescence spectroscopy. The peptides RS20F and RS20CK correspond to CaM-binding amino acid sequence segments of either nonmuscle myosin light chain kinase (nmMLCK) or calmodulin-dependent protein kinase II (CaMPK-II), respectively. Both steady-state and time-resolved fluorescence data were collected from the various peptide-CaM complexes. Steady-state fluorescence intensity measurements indicated that, in the presence of an excess of calcium, both peptides bind to the calmodulin mutants with a 1:1 stoichiometry. The tryptophans located in loops I and IV exhibited red-shifted emission maxima (356 nm), high quantum yields (0.3), and long average lifetimes (6 ns). They responded in a similar manner to peptide binding, by only slight changes in their fluorescence features. In contrast, the fluorescence intensity of the tryptophans in loops II and III decreased markedly, and their fluorescence spectrum was blue-shifted upon peptide binding. Analysis of the tryptophan fluorescence decay of the last mentioned calmodulins supports a model in which the equilibrium between two (Trp-99) or three (Trp-62) states of these tryptophan residues, each characterized by a different lifetime, was altered toward the blue-shifted short lifetime component upon peptide binding. Taken together, these data provide new evidence that both lobes of calmodulin are involved in peptide binding. Both peptides induced similar changes in the fluorescence properties of the tryptophan residues located in the calcium-binding loops, with the exception of calmodulin with Trp-135. For this last mentioned calmodulin, slight differences were observed. Tryptophan in the central helix responded differently to RS20F and RS20CK binding. RS20F binding induced a red-shift in the emission maximum of Trp-81 while RS20CK induced a blue-shift. The quenching rate of Trp-81 by iodide was slightly reduced upon RS20CK binding, while RS20F induced a 2-fold increase. These results provide evidence that the environment of Trp-81 is different in each case and are, therefore, consistent with the hypothesis that the central helix can play a differential role in the recognition of, or response to, CaM-binding structures.     

Optical spectroscopic characterization of single tryptophan mutants of chicken skeletal troponin C: evidence for interdomain interaction.

The effects of metal ion binding on the optical spectroscopic properties and temperature stability of two single tryptophan mutants of chicken skeletal TnC, F78W and F154W, have been examined. The absence of tyrosine and other tryptophan residues allowed the unambiguous assignment of the spectral signal from the introduced Trp residue. Changes in the molar ellipticity values in the far-UV CD spectra of the mutant proteins on metal ion binding were similar to those of wild-type TnC suggesting that the introduction of the Trp residue had no effect on the total secondary structure content. The fluorescence and near-UV absorbance data reveal that, in the apo state, Trp-78 is buried while Trp-154 is exposed to solvent. Additionally, the highly resolved (1)L(b) band of Trp-78 seen in the near-UV absorbance and CD spectra of the apo state of F78W suggest that this residue is likely in a rigid molecular environment. In the calcium-saturated state, Trp-154 becomes buried while the solvent accessibility of Trp-78 increases. The fluorescence emission and near-UV CD of Trp-78 in the N-terminal domain were sensitive to calcium binding at the C-terminal domain sites. Measurements of the temperature stability reveal that events occurring in the N-terminal domain affect the stability of the C-terminal domain and vice versa. This, coupled with the titration data, strongly suggests that there are interactions between the N- and C-terminal domains of TnC.     

Fluorescence analysis of a dynamic loop in the PCAF/GCN5 histone acetyltransferase

PCAF and GCN5 are histone acetyltransferase (HAT) paralogs which play roles in the remodeling of chromatin in health and disease. Previously, a conformationally flexible loop in the catalytic domain had been observed in the X-ray structures of GCN5 in different liganded states. Here, the conformation and dynamics of this PCAF/GCN5 alpha5-beta6 loop was investigated in solution using tryptophan fluorescence. A mutant human PCAF HAT domain (PCAF(Wloop)) was created in which the natural tryptophan (Trp-514) remote from the alpha5-beta6 loop was replaced with tyrosine and a glutamate within the loop (Glu-641) was substituted with tryptophan. This PCAF(Wloop) protein exhibited catalytic parameters within 3-fold of those of the wild-type PCAF catalytic domain, suggesting that the loop mutation was not deleterious for HAT activity. While saturating CoASH induced a 30% quenching of Trp fluorescence in PCAF(Wloop), binding of the high-affinity bisubstrate analogue H3-CoA-20 led to a 2-fold fluorescence increase. These different effects correlate with the different alpha5-beta6 loop conformations seen previously in X-ray structures. On the basis of stopped-flow fluorescence studies, binding of H3-CoA-20 to PCAF(Wloop) proceeds via a rapid association step followed by a slower conformational change involving loop movement. Time-resolved fluorescence measurements support a model in which the alpha5-beta6 loop in the H3-CoA-20-PCAF(Wloop) complex exists in a narrower ensemble of conformations compared to free PCAF(Wloop). The relevance of loop dynamics to PCAF/GCN5 catalysis and substrate specificity are discussed.     

Role of tryptophan residues in interfacial binding of phosphatidylinositol-specific phospholipase C

The phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis exhibits several types of interfacial activation. In the crystal structure of the closely related Bacillus cereus PI-PLC, the rim of the active site is flanked by a short helix B and a loop that show an unusual clustering of hydrophobic amino acids. Two of the seven tryptophans in PI-PLC are among the exposed residues. To test the importance of these residues in substrate and activator binding, we prepared several mutants of Trp-47 (in helix B) and Trp-242 (in the loop). Two other tryptophans, Trp-178 and Trp-280, which are not near the rim, were mutated as controls. Kinetic (both phosphotransferase and cyclic phosphodiesterase activities), fluorescence, and vesicle binding analyses showed that both Trp-47 and Trp-242 residues are important for the enzyme to bind to interfaces, both activating zwitterionic and substrate anionic surfaces. Partitioning of the enzyme to vesicles is decreased more than 10-fold for either W47A or W242A, and removal of both tryptophans (W47A/W242A) yields enzyme with virtually no affinity for phospholipid surfaces. Replacement of either tryptophan with phenylalanine or isoleucine has moderate effects on enzyme affinity for surfaces but yields a fully active enzyme. These results are used to describe how the enzyme is activated by interfaces.     

Interdomain interaction through helices A and B of DnaK peptide binding domain

In order to better define the structural elements involved in allosteric signalling, wild-type DnaK and three deletion mutants of the peptide binding domain have been characterized by biophysical (steady-state and time-resolved fluorescence) and biochemical methods. In the presence of ATP the chemical environment of the single tryptophan residue of DnaK, located in the ATPase domain, becomes less polar, as seen by a blue shift of the emission maximum and a shortening of the fluorescence lifetime, and its accessibility to polar quenchers is drastically reduced. These nucleotide-dependent modifications are also observed for the deletion mutant DnaK1-537, but not for DnaK1-507 or DnaK1-385, and thus rely on the presence of residues 507–537 (helices A and the N-terminal half of B) of the peptide binding domain. These data indicate that αA and half αB contribute to the allosteric communication of DnaK. In the presence of ATP, they promote a conformational change that displaces a residue(s) of the peptide binding domain towards a region of the ATPase domain where the tryptophan residue (W102) is located. A putative role for these helical segments as regulators of the position of the lid is discussed.     

Involvement of a conserved tryptophan residue in the UDP-glucose binding of large clostridial cytotoxin glycosyltransferases

Large clostridial cytotoxins catalyze the glucosylation of Rho/Ras GTPases using UDP-glucose as a cosubstrate. By site-directed mutagenesis of Clostridium sordellii lethal toxin and Clostridium difficile toxin B fragments, we identified tryptophan 102, which is located in a conserved region within the catalytic domain of all clostridial cytotoxins, to be crucial for UDP-glucose binding. Exchange of Trp-102 with alanine decreased the glucosyltransferase activity by about 1,000-fold and blocked cytotoxic activity after microinjection. Replacement of Trp-102 by tyrosine caused a 100-fold reduction in enzyme activity, indicating a partial compensation of the tryptophan function by tyrosine. Decrease in glucosyltransferase and glycohydrolase activity was caused predominantly by an increase in the K(m) for UDP-glucose of these mutants. The data indicate that the conserved tryptophan residue is implicated in the binding of the cosubstrate UDP-glucose by large clostridial cytotoxins. Data bank searches revealed different groups of proteins sharing the recently identified DXD motif (Busch, C., Hofmann, F., Selzer, J., Munro, J., Jeckel, D., and Aktories, K. (1998) J. Biol. Chem. 273, 19566-19572) and a conserved region defined by a tryptophan residue equivalent to Trp-102 of C. sordellii lethal toxin. From our findings, we propose a novel family of glycosyltransferases which includes both prokaryotic and eukaryotic proteins.     

Spatial relationship between the prodan site,Trp-214, and Cys-34 residues in human serum albumin and loss of structure through incremental unfolding

Prodan (6-propionyl-2-(dimethylamino)-naphthalene), a competitive inhibitor of warfarin binding to human serum albumin (HSA) at drug site I, was used to determine the inter- and intradomain distances of HSA. The fluorescence resonance energy transfer (FRET) distances between prodan and Trp-214, prodan and 7-(diethyl amino)-4-methylcoumarin 3-maleimide (CM)-modified Cys-34, and Trp-214 and CM-Cys-34 were determined to be 25.5 +/- 0.5 A, 33.1 +/- 0.8 A, and 32.4 +/- 1 A, respectively. FRET analysis showed that low concentration of palmitic acid (5 microM) increased the interdomain distance between the Trp-214 in domain II and CM-Cys-34 in domain I by approximately 5 A without perturbing the secondary structure of HSA and the immediate environment of Trp-214. Palmitic acid (5 microM) increased the prodan fluorescence by increasing the quantum yield of bound prodan without altering the tryptophan environment. However, palmitic acid (>10 microM) decreased the prodan fluorescence and increased the tryptophan fluorescence. Our results indicate that the high affinity palmitic acid binding site is located at the interface of domains I and II. On the basis of our measurements, a schematic model representing the drug site-1, Trp-214, and Cys-34 along with the palmitic acid sites has been constructed. In addition, prodan fluorescence, FRET, and ligand binding were used to monitor guanidine hydrochloride-induced denaturation of HSA. An analysis of the equilibrium unfolding data suggests that HSA undergoes a two-state unfolding transition with no detectable intermediate. However, kinetic analysis using multiple probes and thermal denaturation studies showed that the unfolding of the prodan site in HSA preceded the unfolding of tryptophan environment. In addition, the separation of domain I and II occurred before the global unfolding of the protein. The data support the idea that HSA loses its structure incrementally during its unfolding.     

Engineered Tet repressor mutants with single tryptophan residues as fluorescent probes. Solvent accessibilities of DNA and inducer binding sites and interaction with tetracycline

Mutants of the Tn10-encoded Tet repressor containing single or no tryptophan residues were constructed by oligonucleotide-directed mutagenesis. The Trp-75 to Phe exchange reduces the dissociation rate of the complex with the inducer tetracycline by a factor of 2. The Trp-43 to Phe exchange has no effect on inducer binding. The fluorescence emission spectra of both tryptophan residues are quenched to a different extent by binding of tetracycline: Trp-75 is quenched to zero and Trp-43 to only 50%. It is concluded that Trp-75 is in the vicinity of the inducer binding site. The different fluorescence emission spectra of both tryptophan residues depend on the native structure of Tet repressor. Quenching studies with iodide indicate that the DNA binding motif is solvent exposed in free repressor and moves towards the interior of the protein upon inducer binding. The inducer binding site is in the interior of the protein. The fluorescence of tetracycline is enhanced upon binding to Tet repressor. The excitation at 280 nm results mainly from the change in environment and in part from energy transfer from tryptophan to the drug.     

The environments of Trp-248 and Trp-330 in tryptophan indole-lyase from Escherichia coli

The two tryptophan residues, Trp-248 and Trp-330, in tryptophan indole-lyase (tryptophanase) from E. coli have been separately mutated to phenylalanine using site-directed mutagenesis. Both single tryptophan mutant enzymes have full catalytic activity, but exhibit different fluorescence and near-UV circular dichroism spectra. These results indicate that Trp-330 is more deeply buried than is Trp-248, and is in a more asymmetric environment. Neither residue reacts with N-bromosuccinimide (NBS), although tryptophan indole-lyase is inactivated by NBS. These results demonstrate that the tryptophan residues in tryptophan indole-lyase are not catalytically essential.     

Use of Akt inhibitor and a drug-resistant mutant validates a critical role for protein kinase B/Akt in the insulin-dependent regulation of glucose and system A amino acid uptake

Protein kinase B (PKB)/Akt has been strongly implicated in the insulin-dependent stimulation of GLUT4 translocation and glucose transport in skeletal muscle and fat cells. Recently an allosteric inhibitor of PKB (Akti) that selectively targets PKBalpha and -beta was reported, but as yet its precise mechanism of action or ability to suppress key insulin-regulated events such as glucose and amino acid uptake and glycogen synthesis in muscle cells has not been reported. We show here that Akti ablates the insulin-dependent regulation of these processes in L6 myotubes at submicromolar concentrations and that inhibition correlates tightly with loss of PKB activation/phosphorylation. Similar findings were obtained using 3T3-L1 adipocytes. Akti did not inhibit IRS1 tyrosine phosphorylation, phosphatidylinositol 3-kinase signaling, or activation of Erks, ribosomal S6 kinase, or atypical protein kinases C but significantly impaired regulation of downstream PKB targets glycogen synthase kinase-3 and AS160. Akti-mediated inhibition of PKB requires an intact kinase pleckstrin homology domain but does not involve suppression of 3-phosphoinositide binding to this domain. Importantly, we have discovered that Akti inhibition is critically dependent upon a solvent-exposed tryptophan residue (Trp-80) that is present within the pleckstrin homology domain of all three PKB isoforms and whose mutation to an alanine (PKB(W80A)) yields an Akti-resistant kinase. Cellular expression of PKB(W80A) antagonized the Akti-mediated inhibition of glucose and amino acid uptake. Our findings support a critical role for PKB in the hormonal regulation of glucose and system A amino acid uptake and indicate that use of Akti and expression of the drug-resistant kinase will be valuable tools in delineating cellular PKB functions.