Labeling of specific lysine residues at the active site of glutamine synthetase

Glutamine synthetase (Escherichia coli) was incubated with three different reagents that react with lysine residues, viz. pyridoxal phosphate, 5'-p-fluorosulfonylbenzoyladenosine, and thiourea dioxide. The latter reagent reacts with the epsilon-nitrogen of lysine to produce homoarginine as shown by amino acid analysis, nmr, and mass spectral analysis of the products. A variety of differential labeling experiments were conducted with the above three reagents to label specific lysine residues. Thus pyridoxal phosphate was found to modify 2 lysine residues leading to an alteration of catalytic activity. At least 1 lysine residue has been reported previously to be modified by pyridoxal phosphate at the active site of glutamine synthetase (Whitley, E. J., and Ginsburg, A. (1978) J. Biol. Chem. 253, 7017-7025). By varying the pH and buffer, one or both residues could be modified. One of these lysine residues was associated with approximately 81% loss in activity after modification while modification of the second lysine residue led to complete inactivation of the enzyme. This second lysine was found to be the residue which reacted specifically with the ATP affinity label 5'-p-fluorosulfonylbenzoyladenosine. Lys-47 has been previously identified as the residue that reacts with this reagent (Pinkofsky, H. B., Ginsburg, A., Reardon, I., Heinrikson, R. L. (1984) J. Biol. Chem. 259, 9616-9622; Foster, W. B., Griffith, M. J., and Kingdon, H. S. (1981) J. Biol. Chem. 256, 882-886). Thiourea dioxide inactivated glutamine synthetase with total loss of activity and concomitant modification of a single lysine residue. The modified amino acid was identified as homoarginine by amino acid analysis. The lysine residue modified by thiourea dioxide was established by differential labeling experiments to be the same residue associated with the 81% partial loss of activity upon pyridoxal phosphate inactivation. Inactivation with either thiourea dioxide or pyridoxal phosphate did not affect ATP binding but glutamate binding was weakened. The glutamate site was implicated as the site of thiourea dioxide modification based on protection against inactivation by saturating levels of glutamate. Glutamate also protected against pyridoxal phosphate labeling of the lysine consistent with this residue being the common site of reaction with thiourea dioxide and pyridoxal phosphate.     

A single negatively charged residue affects the orientation of a membrane protein in the inner membrane of Escherichia coli only when it is located adjacent to a transmembrane domain

The orientation of membrane proteins is determined by the asymmetric distribution of charged residues in the sequences flanking the transmembrane domains. For the inner membrane of Escherichia coli, numerous studies have shown that an excess of positively charged residues defines a cytoplasmic domain of a membrane protein ("positive inside" rule). The role of negatively charged residues in establishing membrane protein topology, however, is not completely understood. To investigate the influence of negatively charged residues on this process in detail, we have constructed a single spanning chimeric receptor fragment comprising the N terminus and first transmembrane domain of the heptahelical G protein-coupled vasopressin V(2) receptor and the first cytoplasmic loop of the beta(2)-adrenergic receptor. When fused to alkaline phosphatase (PhoA), the receptor fragment inserted into the inner membrane of E. coli with its N terminus facing the cytoplasm (N(in)-C(out) orientation), although both membrane-flanking domains had rather similar topogenic determinants. The orientation of the receptor fragment was changed after the introduction of single glutamate residues into the N terminus. Orientation inversion, however, was found to be dependent on the location of the glutamate substitutions, which had to lie within a narrow window up to 6 residues distant from the transmembrane domain. These results demonstrate that a single negatively charged residue can play an active role as a topogenic determinant of membrane proteins in the inner membrane of E. coli, but only if it is located adjacent to a transmembrane domain.     

The conserved histidine 295 does not contribute to proton cotransport by the glutamate transporter EAAC1

Transmembrane glutamate transport by the excitatory amino acid carrier (EAAC1) is coupled to the cotransport of three Na(+) ions and one proton. Previously, we suggested that the mechanism of H(+) cotransport involves protonation of the conserved glutamate residue E373. However, it was also speculated that the cotransported proton is shared in a H(+)-binding network, possibly involving the conserved histidine 295 in the sixth transmembrane domain of EAAC1. Here, we used site-directed mutagenesis together with pre-steady-state electrophysiological analysis of the mutant transporters to test the protonation state of H295 and to determine its involvement in proton transport by EAAC1. Our results show that replacement of H295 with glutamine, an amino acid residue that cannot be protonated, generates a fully functional transporter with transport kinetics that are close to those of the wild-type EAAC1. In contrast, replacement with lysine results in a transporter in which substrate binding and translocation are dramatically inhibited. Furthermore, it is demonstrated that the effect of the histidine 295 to lysine mutation on the glutamate affinity is caused by its positive charge, since wild-type-like affinity can be restored by changing the extracellular pH to 10.0, thus partially deprotonating H295K. Together, these results suggest that histidine 295 is not protonated in EAAC1 at physiological pH and, thus, does not contribute to H(+) cotransport. This conclusion is supported by data from H295C-E373C double mutant transporters which demonstrate that these residues cannot be linked by oxidation, indicating that H295 and E373 are not close in space and do not form a proton binding network. A kinetic scheme is used to quantify the results, which includes binding of the cotransported proton to E373 and binding of a modulatory, nontransported proton to the amino acid side chain in position 295.     

Glutamine as a precursor to N-terminal pyrrolid-2-one-5-carboxylic acid in mouse immunoglobulin λ-type light chains. Amino acid-sequence variability at the N-terminal extra piece of λ-type light-chain precursors

The mRNA molecules coding for three mouse immunoglobulin lambda-type light (L) chains (MOPC-104E lambda(1), RPC-20 lambda(1), MOPC-315 lambda(2)) programme the cell-free synthesis of precursors larger than the mature proteins. Radioactive amino acid-sequence analyses of each of the three precursors labelled with [(3)H]alanine, [(3)H]serine, [(3)H]glutamine, [(3)H]glutamic acid and [(3)H]threonine showed that an extra piece, at least 18 residues long, is linked to the N-terminus of the mature L-chains. The N-terminal extra-peptide segment may be 19 residues long, since analyses of precursors labelled with [(35)S]methionine indicated an additional N-terminal methionine residue which was recovered in low yields. Presumably this is the initiator methionine, which is known to be short lived in eukaryotes. The mature forms of MOPC-104E, RPC-20 and MOPC-315 lambda L-chains are blocked at the N-termini by pyrrolid-2-one-5-carboxylic acid (pyroglutamic acid). Sequence analyses of precursors labelled with [(3)H]glutamine and [(3)H]glutamic acid showed incorporation only of glutamine in a position that matches with the position of pyrrolid-2-one-5-carboxylic acid in the mature forms of all three precursors, and incorporation of glutamic acid in other positions. The data showed the absence of glutamine-glutamic acid interconversion, since the radioactive peaks obtained from either (3)H-labelled amino acid were discrete, and free from cross-contamination. These results prove that glutamine is the precursor amino acid of pyrrolid-2-one-5-carboxylic acid at the N-termini of the mature MOPC-104E lambda(1), RPC-20 lambda(1) and MOPC-315 lambda(2) L-chains. Thus the formation of pyrrolid-2-one-5-carboxylic acid by cyclization of glutamine is a post-translational event which occurs after, or concomitant with, cleavage of the extra piece from the precursor to yield the mature L-chain. The variable (V) regions (110 amino acid residues) of mouse lambda L-chains are quite similar: when compared with that of MOPC-104E lambda(1) chain, the V-region of RPC-20 lambda(1) chain differs in one residue, and the V-region of MOPC-315 lambda(2) chain differs in 11 residues. The partial sequence data show that the N-terminal extra pieces of the two lambda(1) L-chain precursors have, so far, identical partial sequences; the extra piece of the lambda(2) L-chain precursor differs from these in at least three out of 19 positions.     

The role of C-terminal ionic residues in self-association of apolipoprotein A-I

Apolipoprotein A-I (apoA-I) is the main protein of high-density lipoprotein and is comprised of a helical bundle domain and a C-terminal (CT) domain encompassing the last ~65 amino acid residues of the 243-residue protein. The CT domain contains three putative helices (helix 8, 9, and 10) and is critical for initiating lipid binding and harbors sites that mediate self-association of the lipid-free protein. Three lysine residues reside in helix-8 (K195, 206, 208), and three in helix-10 (K226, 238, 239). To determine the role of each CT lysine residue in apoA-I self-association, single, double and triple lysine to glutamine mutants were engineered via site-directed mutagenesis. Circular dichroism and chemical denaturation analysis revealed all mutants retained their structural integrity. Chemical crosslinking and size-exclusion chromatography showed a small effect on self-association when helix-8 lysine residues were changed into glutamine. In contrast, mutation of the three helix-10 lysine residues resulted in a predominantly monomeric protein and K226 was identified as a critical residue. When helix-10 glutamate residues 223, 234, or 235 were substituted with glutamine, reduced self-association was observed similar to that of the helix-10 lysine variants, suggesting ionic interactions between these residues. Thus, helix-10 is a critical part of apoA-I mediating self-association, and disruption of ionic interactions changes apoA-I from an oligomeric state into a monomer. Since the helix-10 triple mutant solubilized phospholipid vesicles at higher rates compared to wild-type apoA-I, this indicates monomeric apoA-I is more potent in lipid binding, presumably because helix-10 is fully accessible to interact with lipids.     

Noncovalent scFv multimers of tumor-targeting anti-Lewis(y) hu3S193 humanized antibody

Single-chain variable fragments (scFvs) of anti-Lewis(y) hu3S193 humanized antibody were constructed by joining the V(H) and V(L) domains with either +2 residues, +1 residue, or by directly linking the domains. In addition two constructs were synthesized in which one or two C-terminal residues of the V(H) domain were removed (-1 residue, -2 residue) and then joined directly to the V(L) domain. An scFv construct in the reverse orientation with the V(L) joined directly to the V(H) domain was also synthesized. Upon transformation into Escherichia coli all scFv constructs expressed active protein. Binding activity, multimeric status, and multivalent properties were assessed by flow cytometry, size exclusion chromatography, and biosensor analysis. The results for hu3S193 scFvs are consistent with the paradigm that scFvs with a linker of +3 residues or more associate to form a non-covalent dimer, and those with a shorter linker or directly linked associate predominantly to form a non-covalent trimer and tetramer that are in equilibrium. While the association of V domains to form either a dimer or trimer/tetramer is governed by the length of the linker, the stability of the trimer/tetramer in the equilibrium mixture is dependent on the affinity of the interaction of the individual V domains to associate to form the larger Fv module.     

The integrity of the ball-and-socket joint between V and C domains is essential for complete activity of a humanized antibody

AF2 is a high affinity murine Ab possessing potent neutralizing activity against human IFN-gamma. In carrying out the modifications to humanize this Ab, we discovered that an initial version displayed affinity for IFN-gamma that was slightly less than that of AF2, but exhibited IFN-gamma-neutralizing activity that was severely diminished. Characterization via site-directed mutagenesis revealed that the majority of this loss in IFN-gamma-neutralizing activity was due to altering the V(H) framework residue at position 11. V(H) position 11 is distal to the binding surface of the Ab; however, it, along with residues 110 and 112, have been identified as forming the socket of a molecular ball-and-socket joint between the V and C domains of the Ig Fab, which influences the elbow angle between these domains. To determine whether disrupting the structure of this joint was the basis for reduced IFN-gamma-neutralizing capacity, we altered residue 148 of C(H1), which with residue 149 comprises the corresponding ball portion of the joint. Changing this single C(H1) domain residue diminished the ability of the Ab to neutralize IFN-gamma to a level similar to that observed with the V(H) alteration. Thus, an intact ball-and-socket joint between the V and C domains in AF2 is required for potent neutralization of IFN-gamma. These results suggest the importance of the elbow angle between Ig V and C domains in Ab activity, and support the hypothesis that this joint can be an important functional element of Ab structure.     

Contributions of CDR3 to V H H domain stability and the design of monobody scaffolds for naive antibody libraries

Camelids produce functional antibodies devoid of light chains. Autonomous heavy chain variable (V(H)H) domains in these molecules have adapted to the absence of the light chain in the following ways: bulky hydrophobic residues replace small aliphatic residues in the former light chain interface, and residues from the third complementarity-determining region (CDR3) pack against the framework and stabilize the global V(H)H domain fold. To determine the specific roles of CDR3 residues in framework stabilization, we used nai;ve phage-displayed libraries, combinatorial alanine-scanning mutagenesis and biophysical characterization of purified proteins. Our results indicate that in the most stable scaffolds, the structural residues in CDR3 reside near the boundaries of the loop and pack against the framework to form a small hydrophobic core. These results allow us to differentiate between structural CDR3 residues that should remain fixed, and CDR3 residues that are tolerant to substitution and can therefore be varied to generate functional diversity within phage-displayed libraries. These methods and insights can be applied to the rapid design of heavy chain scaffolds for the identification of novel ligands using synthetic, antibody-phage libraries. In addition, they shed light on the relationships between CDR3 sequence diversity and the structural stability of the V(H)H domain fold.     

A single amino acid determines lysophospholipid specificity of the S1P1 (EDG1) and LPA1 (EDG2) phospholipid growth factor receptors

The phospholipid growth factors sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are ligands for the related G protein-coupled receptors S1P(1)/EDG1 and LPA(1)/EDG2, respectively. We have developed a model of LPA(1) that predicts interactions between three polar residues and LPA. One of these, glutamine 125, which is conserved in the LPA receptor subfamily (LPA(1)/EDG2, LPA(2)/EDG4, and LPA(3)/EDG7), hydrogen bonds with the LPA hydroxyl group. Our previous S1P(1) study identified that the corresponding glutamate residue, conserved in all S1P receptors, ion pairs with the S1P ammonium. These two results predict that this residue might influence ligand recognition and specificity. Characterization of glutamate/glutamine interchange point mutants of S1P(1) and LPA(1) validated this prediction as the presence of glutamate was required for S1P recognition, whereas LPA recognition was possible with either glutamine or glutamate. The most likely explanation for this dual specificity behavior is a shift in the equilibrium between the acid and conjugate base forms of glutamic acid due to other amino acids surrounding that position in LPA(1), producing a mixture of receptors including those having an anionic glutamate that recognize S1P and others with a neutral glutamic acid that recognize LPA. Thus, computational modeling of these receptors provided valid information necessary for understanding the molecular pharmacology of these receptors.     

Cooperativity in nicotinamide adenine dinucleotide binding induced by mutations of arginine 475 located at the subunit interface in the human liver mitochondrial class 2 aldehyde dehydrogenase

The low-activity Oriental variant of human mitochondrial aldehyde dehydrogenase possesses a lysine rather than a glutamate at residue 487 in the 500 amino acid homotetrameric enzyme. The glutamate at position 487 formed two salt bonds, one to an arginine at position 264 in the same subunit and the other to arginine 475 in a different subunit [Steinmetz, C. G., Xie, P.-G.,Weiner, H., and Hurley, T. D. (1997) Structure 5, 2487-2505]. Mutating arginine 264 to glutamine produced a recombinantly expressed enzyme with nativelike properties; in contrast, mutating arginine 475 to glutamine produced an enzyme that exhibited positive cooperativity in NAD binding. The K(M) for NAD increased 23-fold with a Hill coefficient of 1.8. The binding of both NAD and NADH was affected by the mutation at position 475. Restoring the salt bonds between residues 487 and either or both 264 and 475 did not restore nativelike properties to the Oriental variant. Further, the R475Q mutant was thermally less stable than the native enzyme, Oriental variant, or other mutants. The presence of NAD restored nativelike stability to the mutant. It is concluded that movement of arginine 475 disrupted salt bonds between it and residues other than the one at 487, which caused the apo-R475Q mutant to have properties typical of an enzyme that exhibits positive cooperativity in substrate binding. Breaking the salt bond between glutamate 487 in the Oriental variant and the two arginine residues cannot be the only reason that this enzyme has altered catalytic properties.     

Specificity determining residues in ammonia- and glutamine-dependent carbamoyl phosphate synthetases.

Carbamoyl phosphate synthetases (CPSs) utilize either glutamine or ammonia for the ATP-dependent generation of carbamoyl phosphate. In glutamine-utilizing CPSs (e.g. the single Escherichia coli CPS and mammalian CPS II), the hydrolysis of glutamine to yield ammonia is catalyzed at a triad-type glutamine amidotransferase domain. Non-glutamine-utilizing CPSs (e.g. rat and human CPS I), lacking the catalytic cysteine residue, can generate carbamoyl phosphate only in the presence of free ammonia. Frog CPS I (fCPS I), unlike mammalian CPS Is, retains most of the glutamine amidotransferase residues conserved in glutamine-utilizing CPSs, including an intact catalytic triad, and could therefore be expected to use glutamine. Our work with native fCPS I provides the first demonstration of the inability of this enzyme to bind/utilize glutamine. To determine why fCPS I is unable to utilize glutamine, we compared sequences of glutamine-using and non-glutamine-using CPSs to identify residues that are present or conservatively substituted in all glutamine-utilizing CPSs but absent in fCPS I. We constructed the site-directed mutants Q273E, L270K, Q273E/N240S, and Q273E/L270K in E. coli CPS and have determined that simultaneous occurrence of the two substitutions, Gln-->Glu and Leu-->Lys, found in the frog CPS I glutamine amidotransferase domain are sufficient to eliminate glutamine utilization by the E. coli enzyme.     

The role of interface framework residues in determining antibody V(H)/V(L) interaction strength and antigen-binding affinity

While many antibodies with strong antigen-binding affinity have stable variable regions with a strong antibody heavy chain variable region fragment (V(H))/antibody light chain variable region fragment (V(L)) interaction, the anti-lysozyme IgG HyHEL-10 has a fairly strong affinity, yet a very weak V(H)/V(L) interaction strength, in the absence of antigen. To investigate the possible relationship between antigen-binding affinity and V(H)/V(L) interaction strength, a novel phage display system that can switch two display modes was employed. We focused on the two framework region 2 regions of the HyHEL-10 V(H) and V(L), facing each other at the domain interface, and a combinatorial library was made in which each framework region 2 residue was mixed with that of D1.3, which has a far stronger V(H)/V(L) interaction. The phagemid library, encoding V(H) gene 7 and V(L) amber codon gene 9, was used to transform TG-1 (sup+), and the phages displaying functional variable regions were selected. The selected phages were then used to infect a nonsuppressing strain, and the culture supernatant containing V(H)-displaying phages and soluble V(L) fragment was used to evaluate the V(H)/V(L) interaction strength. The results clearly showed the existence of a key framework region 2 residue (H39) that strongly affects V(H)/V(L) interaction strength, and a marked positive correlation between the antigen-binding affinity and the V(H)/V(L) interaction, especially in the presence of a set of particular V(L) residues. The effect of the H39 mutation on the wild-type variable region was also confirmed by a SPR biosensor as a several-fold increase in antigen-binding affinity owing to an increased association rate, while a slight decrease was observed for the single-chain variable region.     

Two glutamate residues, Glu 208 alpha and Glu 197 beta, are crucial for phosphorylation and dephosphorylation of the active-site histidine residue in succinyl-CoA synthetase.

Succinyl-CoA synthetase catalyzes the reversible reaction succinyl-CoA + NDP + P(i) <--> succinate + CoA + NTP (N denoting adenosine or guanosine). The enzyme consists of two different subunits, designated alpha and beta. During the reaction, a histidine residue of the alpha-subunit is transiently phosphorylated. This histidine residue interacts with Glu 208 alpha at site I in the structures of phosphorylated and dephosphorylated Escherichia coli SCS. We postulated that Glu 197 beta, a residue in the nucleotide-binding domain, would provide similar stabilization of the histidine residue during the actual phosphorylation/dephosphorylation by nucleotide at site II. In this work, these two glutamate residues have been mutated individually to aspartate or glutamine. Glu 197 beta has been additionally mutated to alanine. The mutant proteins were tested for their ability to be phosphorylated in the forward or reverse direction. The aspartate mutant proteins can be phosphorylated in either direction, while the E208 alpha Q mutant protein can only be phosphorylated by NTP, and the E197 beta Q mutant protein can only be phosphorylated by succinyl-CoA and P(i). These results demonstrate that the length of the side chain at these positions is not critical, but that the charge is. Most significantly, the E197 beta A mutant protein could not be phosphorylated in either direction. Its crystal structure shows large differences from the wild-type enzyme in the conformation of two residues of the alpha-subunit, Cys 123 alpha-Pro 124 alpha. We postulate that in this conformation, the protein cannot productively bind succinyl-CoA for phosphorylation via succinyl-CoA and P(i).     

Site-directed mutagenesis and 1H NMR spectroscopy of an interdomain segment in the pyruvate dehydrogenase multienzyme complex of Escherichia coli

Deletion of two of the three homologous lipoyl domains that form the N-terminal half of each dihydrolipoamide acetyltransferase (E2p) polypeptide chain of the Escherichia coli pyruvate dehydrogenase complex can be achieved by in vitro deletion in the structural gene aceF. A site-directed mutagenesis of this shortened aceF gene was carried out to replace the glutamine residue at position 291 (wild-type numbering) with a histidine residue. Residue 291 is near the middle of a long segment (about 30 amino acid residues) of polypeptide chain, rich in alanine, proline, and charged amino acids, that links the remaining lipoyl domain to the dihydrolipoamide dehydrogenase (E3) binding domain in the E2p chain. A fully active enzyme complex was still assembled, and despite the enormous size of the particle (Mr approximately 4 x 10(6)), sharp resonances attributable to the single new histidine residue per E2p chain could be detected in the 400-MHz 1H NMR spectrum of the complex. The sharpness of these resonances, their chemical shifts (7.94 and 7.05 ppm), and the apparent pKa (6.4) of the side chain were all consistent with this histidine residue being exposed to solvent in a conformationally flexible region of the E2p polypeptide chain. These experiments provide direct proof for the conformational flexibility of this region of polypeptide chain, which is thought to play an important part in the movement of the lipoyl domain required for active site coupling in the enzyme complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of HEL4, a soluble, refoldable human V(H) single domain with a germ-line scaffold

The antigen binding site of antibodies usually comprises associated heavy (V(H)) and light (V(L)) chain variable domains, but in camels and llamas, the binding site frequently comprises the heavy chain variable domain only (referred to as V(HH)). In contrast to reported human V(H) domains, V(HH) domains are well expressed from bacteria and yeast, are readily purified in soluble form and refold reversibly after heat-denaturation. These desirable properties have been attributed to highly conserved substitutions of the hydrophobic residues of V(H) domains, which normally interact with complementary V(L) domains. Here, we describe the discovery and characterisation of an isolated human V(H) domain (HEL4) with properties similar to those of V(HH) domains. HEL4 is highly soluble at concentrations of > or =3 mM, essentially monomeric and resistant to aggregation upon thermodenaturation at concentrations as high as 56 microM. However, in contrast to V(HH) domains, the hydrophobic framework residues of the V(H):V(L) interface are maintained and the only sequence changes from the corresponding human germ-line segment (V3-23/DP-47) are located in the loops comprising the complementarity determining regions (CDRs). The crystallographic structure of HEL4 reveals an unusual feature; the side-chain of a framework residue (Trp47) is flipped into a cavity formed by Gly35 of CDR1, thereby increasing the hydrophilicity of the V(H):V(L) interface. To evaluate the specific contribution of Gly35 to domain properties, Gly35 was introduced into a V(H) domain with poor solution properties. This greatly enhanced the recovery of the mutant from a gel filtration matrix, but had little effect on its ability to refold reversibly after heat denaturation. Our results confirm the importance of a hydrophilic V(H):V(L) interface for purification of isolated V(H) domains, and constitute a step towards the design of isolated human V(H) domains with practical properties for immunotherapy.     

Molecular cloning and characterization of a large subunit of Salmonella typhimurium glutamate synthase (GOGAT) gene in Escherichia coli

Two pathways of ammonium assimilation and glutamate biosynthesis have been identified in microorganisms. One pathway involves the NADP-linked glutamate dehydrogenase, which catalyzes the amination of 2-oxoglutarate to form glutamate. An alternative pathway involves the combined activities of glutamine synthetase, which aminates glutamate to form glutamine, and glutamate synthase, which transfers the amide group of glutamine to 2-oxoglutarate to yield two molecules of glutamate. We have cloned the large subunit of the glutamate synthase (GOGAT) from Salmonella typhimurium by screening the expression of GOGAT and complementing the gene in E. coli GOGAT large subunit-deficient mutants. Three positive clones (named pUC19C12, pUC19C13 and pUC19C15) contained identical Sau3AI fragments, as determined by restriction mapping and Southern hybridization, and expressed GOGAT efficiently and constitutively using its own promoter in the heterologous host. The coding region expressed in Escherichia coli was about 170 kDa on SDS-PAGE. This gene spans 4,732 bases, contains an open reading frame of 4,458 nucleotides, and encodes a mature protein of 1,486 amino acid residues (Mr = 166,208). The FMN-binding domain of GOGAT contains 12 glycine residues, and the 3Fe-4S cluster has 3 cysteine residues. The comparison of the translated amino acid sequence of the Salmonella GOGAT with sequences from other bacteria such as Escherichia coli, Salmonella enterica, Shigella flexneri, Yersinia pestis, Vibrio vulnificus and Pseudomonas aeruginosa shows sequence identity between 87 and 95%.     

Asparagine362 is essential for zinc binding and catalysis in the peptidase reaction of Saccharomyces cerevisiae leukotriene A4 hydrolase

Zinc metallopeptidases are ubiquitous enzymes with diverse cellular functions that can be found in most organisms. Leukotriene A₄ hydrolase (LTA4H; E.C. 3.3.2.6) is an unusual zinc metallopeptidase of the M1 family that also possesses an epoxide hydrolase activity; however, the role of its peptidase activity remains unknown. To further characterize the peptidase activity of LTA4H and other closely related metallopeptidases, a multiple sequence alignment and predicted structure were used to target three amino acid residues of yeast LTA4H for mutagenesis: Asn362, Trp365, and Asp399. Although mutating Trp365 and Asp399 had little effect on catalysis, altering Asn362 had varying effects on catalysis, depending on the replacement residue. Mutation of Asn362 to glutamine (N362Q) caused minor catalytic defects, while mutation to leucine (N362L) or glutamate (N362E) caused large reductions in activity. Both N362L and N362E also exhibited an altered pH dependence of catalysis, reduced chloride activation, and reduced zinc affinity and content, indicating that Asn362 may interact with the nearby zinc coordinating residue His344, and possibly with Glu363 as well, to polarize and/or orient these residues.     

Evidence for essential histidines in human pituitary glutaminyl cyclase

Glutaminyl cyclase (QC, EC 2.3.2.5) catalyzes the formation of the pyroglutamyl residue present at the amino terminus of numerous secretory peptides and proteins. Treatment with diethyl pyrocarbonate inactivated recombinant human QC with the apparent modification of three essential histidine residues. Comparisons of the protein sequences of QC from a variety of eukaryotic species show four completely conserved histidine residues. Mutation of each of these residues to glutamine resulted in two mutant enzymes that were inactive (H140Q and H330Q), suggesting a role in catalysis, and two that exhibited increased Km values (H307Q and H319Q), suggesting a role in substrate binding. Consistent with these results is the prediction that QC possesses a zinc aminopeptidase domain in which the four histidines identified here are present in the active site. Mammalian glutaminyl cyclases may, therefore, have structural and catalytic similarities to a family of bacterial zinc aminopeptidases.     

A K52Q substitution in the globular domain of histone H1t modulates its nucleosome binding properties

A comparison of the globular domain sequences of the somatic H1d and testis-specific H1t revealed a single substitution of lysine 52 in H1d to glutamine 54 in H1t, which is one of the three crucial residues within the second DNA binding site. The globular domains of both histones were modeled using the crystal structure of chicken GH5 as a template and was also docked onto the nucleosome structure. The glutamine residue in histone H1t forms a hydrogen bond with main chain carbonyl of methionine-52 (in H1t) and is spatially oriented away from the nucleosome dyad axis. A consequence of this change was a lower affinity of recombinant histone H1t towards Four-way junction DNA and reconstituted 5S mononucleosomes. When Gln-54 in Histone H1t was mutated to lysine, its binding affinity towards DNA substrates was comparable to that of histone H1d. The differential binding of histones H1d and H1t towards reconstituted mononucleosomes was also reflected in the chromatosome-stop assay.     

Studies of the mechanism of glutamine synthetase utilizing pH-dependent behavior in catalysis and binding

The pKa values of enzyme groups of Escherichia coli glutamine synthetase which affect catalysis and/or substrate binding were determined by measuring the pH dependence of Vmax and V/K. Analysis of these data revealed that two enzyme groups are required for catalysis with apparent pKa values of approximately 7.1 and 8.2. The binding of ATP is essentially independent of pH in the range studied while the substrate ammonia must be deprotonated for the catalytic reaction. Using methylamine and hydroxylamine in place of ammonia, the pKa value of the deprotonated amine substrate as expressed in the V/K profiles was shifted to a lower pKa value for hydroxylamine and a higher pKa value for methylamine. These data indicate that the amine substrate must be deprotonated for binding. Hydroxylamine is at least as good a substrate as ammonia judged by the kinetic parameters whereas methylamine is a poor substrate as expressed in both the V and V/K values. Glutamate binding was determined by monitoring fluorescence changes of the enzyme and the data indicate that a protonated residue (pKa = 8.3 +/- 0.2) is required for glutamate binding. Chemical modification by reductive methylation with HCHO indicated that the group involved in glutamate binding most likely is a lysine residue. In addition, the Ki value for the transition state analog, L-3-amino-3-carboxy-propanesulfonamide was measured as a function of pH and the results indicate that an enzyme residue must be protonated (pKa = 8.2 +/- 0.1) to assist in binding. A mechanism for the reaction catalyzed by glutamine synthetase is proposed from the kinetic data acquired herein. A salt bridge is formed between the gamma-phosphate group of ATP and an enzyme group prior to attack by the gamma-carboxyl of glutamate on ATP to form gamma-glutamyl phosphate. The amine substrate subsequently attacks gamma-glutamyl phosphate resulting in formation of the tetrahedral adduct before phosphate release. A base on the enzyme assists in the deprotonation of ammonia during its attack on gamma-glutamyl phosphate or after the protonated carbinol amine is formed. Based on the kinetic data with the three amine substrates, catalysis is not rate-limiting through the pH range 6-9.