Nucleophile in the active site of Escherichia coli galactose-1-phosphate uridylyltransferase: degradation of the uridylyl-enzyme intermediate to N3-phosphohistidine.

The [32P]uridylyl-enzyme intermediate form of Escherichia coli galactose-1-P uridylyltransferase can be converted to a [32P]phosphoryl-enzyme by first cleaving the ribosyl ring with NaIO4 and then heating at pH 10.5 and 50 degrees C for 1 h. After alkaline hydrolysis of the [32P]phosphoryl-enzyme the major radioactive product is N3-[32P]phosphohistidine. A lesser amount of 32Pi is also produced as a side product of the hydrolysis of N3-[32P]phosphohistidine. No N1-phosphohistidine, N-phospholysine, or phosphoarginine can be detected in these hydrolysates. It is concluded that the nucleophile in galactose-1-P uridylyltransferase to which the uridylyl group is bonded in the uridylyl-enzyme intermediate is imidazole N3 of a histidine residue. This degradation procedure should have general applicability in the degradation and characterization of nucleotidyl-proteins.     

Galactose-1-phosphate uridylyltransferase. Purification of the enzyme and stereochemical course of each step of the double-displacement mechanism

A convenient new procedure for purifying galactose-1-phosphate uridylyltransferase from Escherichia coli is described. It departs from earlier methods by introducing the use of a Cibacron Blue-agarose (Bio-Rad Affi-Gel Blue) at an early stage. Purification is completed by ion-exchange chromatography using DEAE-Sephadex A-50. The procedure is substantially shorter than earlier methods and reproducibly yields enzyme of high specific activity suitable for use in structural work such as characterization of the intermediate uridylyl-enzyme. The first step of the galactose-1-P uridylyltransferase reaction is the transfer of the uridylyl group from UDP-glucose to N3 of a histidine residue in the enzyme to form the covalent uridylyl-enzyme and glucose-1-P. The uridylyl-enzyme intermediate then reacts in a second step with galactose-1-P to form UDP-galactose. The enzyme accepts (RP)-UDP alpha S-glucose as a good substrate, converting it to (RP)-UDP alpha S-galactose, i.e., with overall retention of configuration. In this paper we show that reaction of the enzyme with (RP)-[2-14C]UDP alpha S-glucose produces a [2-14C]uridylyl alpha S-enzyme that can be converted by base-catalyzed cyclization to (RP)-[2-14C]cUMPS. Inasmuch as cyclization must have proceeded with inversion of configuration at phosphorus, the corresponding configuration in the intermediate must have been the inverse of that in the substrate. Therefore, formation of uridylyl alpha S-enzyme from (RP)-UDP alpha S-glucose proceeds with inversion of configuration, and overall retention arises from inversion in each of the two steps. The results support the authenticity of the isolated uridylyl-enzyme as the true reaction intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Roles of histidine-103 and tyrosine-235 in the function of the prolipoprotein diacylglyceryl transferase of Escherichia coli.

Phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) is the first enzyme in the posttranslational sequence of reactions resulting in the lipid modification of lipoproteins in bacteria. A previous comparison of the primary sequences of the Lgt enzymes from phylogenetically distant bacterial species revealed several highly conserved amino acid sequences throughout the molecule; the most extensive of these was the region 103HGGLIG108 in the Escherichia coli Lgt (H.-Y. Qi, K. Sankaran, K. Gan, and H. C. Wu, J. Bacteriol. 177:6820-6824, 1995). These studies also revealed that the kinetics of inactivation of E. coli Lgt with diethylpyrocarbonate were consistent with the modification of a single essential histidine or tyrosine residue. The current study was conducted in an attempt to identify this essential amino acid residue in order to further define structure-function relationships in Lgt. Accordingly, all of the histidine residues and seven of the tyrosine residues of E. coli Lgt were altered by site-directed mutagenesis, and the in vitro activities of the altered enzymes, as well the abilities of the respective mutant lgt alleles to complement the temperature-sensitive phenotype of E. coli SK634 defective in Lgt activity, were determined. The data obtained from these studies, in conjunction with additional chemical inactivation studies, support the conclusion that His-103 is essential for Lgt activity. These studies also indicated that Tyr-235 plays an important role in the function of this enzyme. Although other histidine and tyrosine residues were not found to be essential for Lgt activity, alterations of His-196 resulted in a significant reduction of in vitro activity.     

The roles of Glu-327 and His-446 in the bisphosphatase reaction of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase probed by NMR spectroscopic and mutational analyses of the enzyme in the transient phosphohistidine intermediate complex

The bisphosphatase domain derived from the rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was studied by 1H-13C HMQC NMR spectroscopy of the histidine C2' and H2' nuclei. The bacterially expressed protein was specifically labeled with 13C at the ring C2' position of the histidines. Each of the seven histidine residues gave rise to a single cross-peak in the HMQC spectra, and these were assigned by use of a series of histidine-to-alanine point mutants. His-304, His-344, and His-469 exhibit 13C and 1H resonances that titrated with pH, while the remaining histidine-associated resonances did not. The 13C and 1H chemical shifts indicate that at neutral pH, His-304 and His-446 are deprotonated, while His-469 is protonated. The pKa of His-344 was determined to be 7.04. The 13C chemical shifts suggest that the deprotonated His-258 exists as the N1' tautomer, while His-392 and His-419 are protonated in the resting, wild-type enzyme. Mutation of the remaining member of the catalytic triad, Glu-327, to alanine in the resting enzyme caused an upfield shift of 1.58 and 1.30 ppm in the 1H and 13C dimensions, respectively, and significant narrowing of the His-258 cross-peak. Mutation of His-446 to alanine produced perturbations of the His-258 cross-peak that were similar to those detected in the E327A mutant. The His-392 resonances were also shifted by the E327A and H446A mutations. These observations strongly suggest that residues His-258, Glu-327, His-392, and His-446 exist within a network of interacting residues that encompasses the catalytic site of the bisphosphatase and includes specific contacts with the C-terminal regulatory region of the enzyme. The specifically 13C-labeled bisphosphatase was monitored during turnover by HMQC spectra acquired from the transient N3' phosphohistidine intermediate complex in the wild-type enzyme, the E327A mutant, and the H446A mutant. These complexes were formed during reaction with the physiological substrate fructose-2, 6-bisphosphate. Upon formation of the phosphohistidine at His-258, the 13C and 1H resonances of this residue were shifted downfield by 1.7 and 0.31 ppm, respectively, in the wild-type enzyme. The upfield shifts of the His-258 resonances in the E327A and H446A mutant resting enzymes were reversed when the phosphohistidine was formed, generating spectra very similar to that of the wild-type enzyme in the intermediate complex. In contrast, the binding of fructose-6-phosphate, the reaction product, to the resting enzyme did not promote significant changes in the histidine-associated resonances in either the wild-type or the mutant enzymes. The interpretation of these data within the context of the X-ray crystal structures of the enzyme is used to define the role of Glu-327 in the catalytic mechanism of the bisphosphatase and to identify His-446 as a putative link in the chain of molecular events that results in activation of the bisphosphatase site by cAMP-dependent phosphorylation of the hepatic bifunctional enzyme.     

Galactose-1-phosphate uridylyltransferase: isolation and properties of a uridylyl-enzyme intermediate.

Galactose-1-P uridylyltransferase catalyzes the interconversion of UDP-galactose and galactose-1-P with UDP-galactose and glucose-1-P by a double displacement pathway involving a uridylyl-enzyme intermediate. The amount of radioactivity incorporated into the protein by uracil-labeled UDP-glucose is decreased by the presence of UDP-galactose, which completes with UDP-glucose for uridylylating the enzyme. The amount of glucose-1-P released upon reaction of the enzyme with UDP-glucose indicates that the dimeric enzyme contains more than one active site per molecule, 1.7 on the average for the most active preparation obtained. This suggests that there is one uridylylation site per subunit and that the subunits are similar or identical. The ureidylyl-enzyme is stable to mild alkaline conditions, 0.10 M NaOH at 60 degrees C for 1 h, but is very sensitive to acid, being largely hydrolyzed after 12 h at pH 3.5 and 4 degrees C. The principal radioactive product resulting from hydrolysis of [uracil-2-14C]uridylyl-ens of the uridylyl-enzyme under the latter conditions is [l]ump. The hydrolytic properties of the uridylyl-enzyme show that the uridylyl moiety is bonded to the protein through a phosphoramidate linkage. Complementary studies on the effects of group selective reagents on the activity of the enzyme suggest that the active site nucleophile to which the uridylyl group is bonded may be a histidine residue. The enzyme is rapidly inactivated by diethyl pyrocarbonate at pH 6 and 0 degrees C and reactivated by NH2OH. UDP-glucose at 0.5 mM fully protects the enzyme against diethyl pyrocarbonate while 70 mM galactose-1-P has only a slight protective effect. Uridylyl-enzyme in inactivated by diethyl pyrocarbonate at no more than 2% of the rate for free enzyme. The enzyme is not inactivated by NaBH4 or by NaBH4 in the presence of UDP-glucose. It is not inhibited by 1 mM pyridoxal phosphate or by 0.5 mM 5-nitrosalicylaldehyde at pH 8.6 and it is not inactivated by NaBH4 in the presence of pyridoxal phosphate. The enzyme is inactivated by 5 to 50 muM p-hydroxymercuribenzoate at pH 8.5, but substrates exert no detectable protective effect against this reagent. It is concluded that the enzyme contains at least one essential sulfhydryl group which is not located in the active site in such a way as to be shielded by substrates.     

Role of aspartate-133 and histidine-458 in the mechanism of tryptophan indole-lyase from Proteus vulgaris

Tryptophan indole-lyase (Trpase) from Proteus vulgaris is a pyridoxal 5'-phosphate dependent enzyme that catalyzes the reversible hydrolytic cleavage of L-Trp to yield indole and ammonium pyruvate. Asp-133 and His-458 are strictly conserved in all sequences of Trpase, and they are located in the proposed substrate-binding region of Trpase. These residues were mutated to alanine to probe their role in substrate binding and catalysis. D133A mutant Trpase has no measurable activity with L-Trp as substrate, but still retains activity with S-(o-nitrophenyl)-L-cysteine, S-alkyl-L-cysteines, and beta-chloro-L-alanine. H458A mutant Trpase has 1.6% of wild-type Trpase activity with L-Trp, and high activity with S-(o-nitrophenyl)-L-cysteine, S-alkyl-L-cysteines, and beta-chloro-L-alanine. H458A mutant Trpase does not exhibit the pK(a) of 5.3 seen in the pH dependence of k(cat)/K(m) of L-Trp for wild-type Trpase. Both mutant enzymes are inhibited by L-Ala, L-Met, and L-Phe, with K(i) values similar to those of wild-type Trpase, but oxindolyl-L-alanine and beta-phenyl-DL-serine show much weaker binding to the mutant enzymes, suggesting that Asp-133 and His-458 are involved in the binding of these ligands. D133A and H458A mutant Trpase exhibit absorption and CD spectra in the presence of substrates and inhibitors that are similar to wild-type Trpase, with peaks at about 420 and 500 nm. The rate constants for formation of the 500 nm bands for the mutant enzymes are equal to or greater than those of wild-type Trpase, indicating that Asp-133 and His-458 do not play a role in the formation of quinonoid intermediates. In constrast to wild-type and H458A mutant Trpase, D133A mutant Trpase forms an intermediate from S-ethyl-L-Cys that absorbs at 345 nm, and is likely to be an alpha-aminoacrylate. Crystals of D133A and H458A mutant Trpase bind amino acids with similar affinity as the proteins in solution, except for L-Ala, which binds to D133A mutant Trpase crystals about 20-fold stronger than in solution. These results suggest that Asp-133 and His-458 play an important role in the elimination reaction of L-Trp. Asp-133 likely forms a hydrogen bond directly to the indole NH of the substrate, while His-458 probably is hydrogen bonded to Asp-133.     

Lactose transport system of Streptococcus thermophilus. The role of histidine residues.

The lactose transport protein (LacS) of Streptococcus thermophilus is a chimeric protein consisting of an amino-terminal carrier domain and a carboxyl-terminal phosphoenolpyruvate:sugar phosphotransferase system (PTS) IIA protein domain. The histidine residues of LacS were changed individually into glutamine or arginine residues. Of the 11 histidine residues present in LacS, only the His-376 substitution in the carrier domain significantly affected sugar transport. The region around His-376 was found to exhibit sequence similarity to the region around His-322 of the lactose transport protein (LacY) of Escherichia coli, which has been implicated in sugar binding and in coupling of sugar and H+ transport. The H376Q mutation resulted in a reduced rate of uptake and altered affinity for lactose (beta-galactoside), melibiose (alpha-galactoside), and the lactose analog methyl-beta-D-thiogalactopyranoside. Similarly, the extent of accumulation of the galactosides by cells expressing LacS(H376Q) was highly reduced in comparison to cells bearing the wild-type protein. Nonequilibrium exchange of lactose and methyl-beta-D-thiogalactopyranoside by the H376Q mutant was approximately 2-fold reduced in comparison to the activity of the wild-type transport protein. The data indicate that His-376 is involved in sugar recognition and is important, but not essential, for the cotransport of protons and galactosides. The carboxyl-terminal domain of LacS contains 2 histidine residues (His-537 and His-552) that are conserved in seven homologous IIA protein(s) (domains) of PTSs. P-enolpyruvate-dependent phosphorylation of wild-type LacS, but not of the mutant H552Q, was demonstrated using purified Enzyme I and HPr, the general energy coupling proteins of the PTS, and inside-out membrane vesicles isolated from E. coli in which the lactose transport gene was expressed. The His-537 and His-552 mutations did not affect transport activity when the corresponding genes were expressed in E. coli.     

Alternative proton donors/acceptors in the catalytic mechanism of the glutathione reductase of Escherichia coli: the role of histidine-439 and tyrosine-99

The cloned Escherichia coli gor gene encoding the flavoprotein glutathione reductase was placed under the control of the tac promoter in the plasmid pKK223-3, allowing expression of glutathione reductase at levels approximately 40,000 times those of untransformed cells. This greatly facilitated purification of the enzyme. By directed mutagenesis of the gor gene, His-439 was changed to glutamine (H439Q) and alanine (H439A). The tyrosine residue at position 99 was changed to phenylalanine (Y99F), and in another experiment, the H439Q and Y99F mutations were united to form the double mutant Y99FH439Q. His-439 is thought to act in the catalytic mechanism as a proton donor/acceptor in the glutathione-binding pocket. The H439Q and H439A mutants retain approximately 1% and approximately 0.3%, respectively, of the catalytic activity of the wild-type enzyme. This reinforces our previous finding [Berry et al. (1989) Biochemistry 28, 1264-1269] that direct protonation and deprotonation of the histidine residue are not essential for the reaction to occur. The retention of catalytic activity by the H439A mutant demonstrates further that a side chain capable of hydrogen bonding to a water molecule, which might then act as proton donor, also is not essential at this position. Tyr-99 is a further possible proton donor in the glutathione-binding pocket, but the Y99F mutant was essentially fully active, and the Y99FH439Q double mutant also retained approximately 1% of the wild-type specific activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of the four conserved histidine residues in the amidotransferase domain of carbamoyl phosphate synthetase

Carbamoyl phosphate synthetase from Escherichia coli catalyzes the formation of carbamoyl phosphate from ATP, bicarbonate, and glutamine. The amidotransferase activity of this enzyme is catalyzed by the smaller of the two subunits of the heterodimeric protein. The roles of four conserved histidine residues within this subunit were probed by site-directed mutagenesis to asparagine. The catalytic activities of the H272N and H341N mutants are not significantly different than that of the wild-type enzyme. The H353N mutant is unable to utilize glutamine as a nitrogen source in the synthetase reaction or the partial glutaminase reaction. However, binding to the glutamine active site is not impaired in the H353N enzyme since glutamine is found to activate the partial ATPase reaction by 40% with a Kd of 54 microM. The H312N mutant has a Michaelis constant for glutamine that is 2 orders of magnitude larger than the wild-type value, but the maximal rate of glutamine hydrolysis is unchanged. These results are consistent with His-353 functioning as a general acid/base catalyst for proton transfers while His-312 serves a critical role for the binding of glutamine to the active site.     

Acid-base catalysis in the extradiol catechol dioxygenase reaction mechanism: site-directed mutagenesis of His-115 and His-179 in Escherichia coli 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB)

The extradiol catechol dioxygenases catalyze the non-heme iron(II)-dependent oxidative cleavage of catechols to 2-hydroxymuconaldehyde products. Previous studies of a biomimetic model reaction for extradiol cleavage have highlighted the importance of acid-base catalysis for this reaction. Two conserved histidine residues were identified in the active site of the class III extradiol dioxygenases, positioned within 4-5 A of the iron(II) cofactor. His-115 and His-179 in Escherichia coli 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) were replaced by glutamine, alanine, and tyrosine. Each mutant enzyme was catalytically inactive for extradiol cleavage, indicating the essential nature of these acid-base residues. Replacement of neighboring residues Asp-114 and Pro-181 gave D114N, P181A, and P181H mutant enzymes with reduced catalytic activity and altered pH/rate profiles, indicating the role of His-179 as a base and His-115 as an acid. Mutant H179Q was catalytically active for the lactone hydrolysis half-reaction, whereas mutant H115Q was inactive, implying a role for His-115 in lactone hydrolysis. A catalytic mechanism involving His-179 and His-115 as acid-base catalytic residues is proposed.     

lac permease of Escherichia coli: histidine-205 and histidine-322 play different roles in lactose/H+ symport

The lac permease of Escherichia coli was modified by site-directed mutagenesis such that His-205 or His-322 is replaced with either Asn or Gln. Permease with Asn or Gln in place of His-205 exhibits normal activity, while permease with Asn or Gln in place of His-322 exhibits no activity. The results are consistent with the interpretation that His-205 and His-322 play different roles in lactose/H+ symport, the former involving hydrogen bonding of the imidazole nitrogens and the latter requiring positive charge in the imidazole ring. In addition, it is demonstrated that permease with Arg in place of His-322 does not catalyze efflux, exchange, or counterflow. The observations, in conjunction with those in the accompanying paper [Carrasco, N., Antes, L. M., Poonian, M. S., & Kaback, H. R. (1986) Biochemistry (following paper in this issue)], suggest that His-322 plays an important role in H+ translocation, possibly as a component of a charge-relay system with Glu-325, a neighboring residue in helix 10.     

Oxygen-dependent coproporphyrinogen III oxidase (HemF) from Escherichia coli is stimulated by manganese

During heme biosynthesis in Escherichia coli two structurally unrelated enzymes, one oxygen-dependent (HemF) and one oxygen-independent (HemN), are able to catalyze the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX. Oxygen-dependent coproporphyrinogen III oxidase was produced by overexpression of the E. coli hemF in E. coli and purified to apparent homogeneity. The dimeric enzyme showed a Km value of 2.6 microm for coproporphyrinogen III with a kcat value of 0.17 min-1 at its optimal pH of 6. HemF does not utilize protoporphyrinogen IX or coproporphyrin III as substrates and is inhibited by protoporphyrin IX. Molecular oxygen is essential for the enzymatic reaction. Single turnover experiments with oxygen-loaded HemF under anaerobic conditions demonstrated electron acceptor function for oxygen during the oxidative decarboxylation reaction with the concomitant formation of H2O2. Metal chelator treatment inactivated E. coli HemF. Only the addition of manganese fully restored coproporphyrinogen III oxidase activity. Evidence for the involvement of four highly conserved histidine residues (His-96, His-106, His-145, and His-175) in manganese coordination was obtained. One catalytically important tryptophan residue was localized in position 274. None of the tested highly conserved cysteine (Cys-167), tyrosine (Tyr-135, Tyr-160, Tyr-170, Tyr-213, Tyr-240, and Tyr-276), and tryptophan residues (Trp-36, Trp-123, Trp-166, and Trp-298) were found important for HemF activity. Moreover, mutation of a potential nucleotide binding motif (GGGXXTP) did not affect HemF activity. Two alternative routes for HemF-mediated catalysis, one metal-dependent, the other metal-independent, are proposed.     

Covalent heterogeneity of the human enzyme galactose-1-phosphate uridylyltransferase

Galactose-1-phosphate uridylyltransferase (GALT) acts by a double displacement mechanism, catalyzing the second step in the Leloir pathway of galactose metabolism. Impairment of this enzyme results in the potentially lethal disorder, galactosemia. Although the microheterogeneity of native human GALT has long been recognized, the biochemical basis for this heterogeneity has remained obscure. We have explored the possibility of covalent GALT heterogeneity using denaturing two-dimensional gel electrophoresis and Western blot analysis to fractionate and visualize hemolysate hGALT, as well as the human enzyme expressed in yeast. In both contexts, two predominant GALT species were observed. To define the contribution of uridylylated enzyme intermediate to the two-spot pattern, we exploited the null allele, H186G-hGALT. The Escherichia coli counterpart of this mutant protein (H166G-eGALT) has previously been demonstrated to fold properly, although it cannot form covalent intermediate. Analysis of the H186G-hGALT protein demonstrated a single predominant species, implicating covalent intermediate as the basis for the second spot in the wild-type pattern. In contrast, three naturally occurring mutations, N314D, Q188R, and S135L-hGALT, all demonstrated the two-spot pattern. Together, these data suggest that uridylylated hGALT comprises a significant fraction of the total GALT enzyme pool in normal human cells and that three of the most common patient mutations do not disrupt this distribution.     

Two distinct proton donors at the active site of Escherichia coli 2,4-dienoyl-CoA reductase are responsible for the formation of different products

NADPH-dependent 2,4-dienoyl-CoA reductase (DCR) is one of the auxiliary enzymes required for the beta-oxidation of unsaturated fatty acids. Mutants of Escherichia coli DCR were generated by site-directed mutagenesis to explore the molecular mechanism of this enzyme. The Tyr166Phe mutant, which was expected to be inactive due to the loss of its putative proton donor residue, exhibited 27% of the wild-type activity. However, the product of the reduction was 3-enoyl-CoA instead of 2-enoyl-CoA, the normal product. Glu164 seems to function as proton donor in the Tyr166Phe mutant, because the Tyr166Phe/ Glu164Gln double mutant was inactive whereas the Glu164Ala mutant exhibited low but significant activity. His252 is important for the efficient operation of Tyr166 because a His252Ala mutation by itself reduced the activity of DCR by 3 orders of magnitude, whereas the Tyr166Phe/His252Ala double mutation exhibited 4.4% of the wild-type activity. This data supports a mechanism that has Tyr166 with the assistance of His252 acting as proton donor in the wild-type enzyme to produce 2-enoyl-CoA, whereas Glu164 serves as the proton donor in the absence of Tyr166 to yield 3-enoyl-CoA. A Cys337Ala mutation, which resulted in the loss of most of the iron and acid-labile sulfur, decreased the reductase activity more than 1000-fold. This observation agrees with the proposed operation of an intramolecular electron transport chain that is essential for the effective catalysis of E. coli DCR.     

Site-directed mutagenesis of histidine-13 and histidine-114 of human angiogenin. Alanine derivatives inhibit angiogenin-induced angiogenesis

The roles of His-13 and His-114 in the ribonucleolytic and angiogenic activities of human angiogenin have been investigated by site-directed mutagenesis. Replacement of either residue by alanine (H13A and H114A) decreases enzymatic activity toward tRNA by at least 10,000-fold and virtually abolishes 10,000-fold and virtually abolishes angiogenic activity in the chick embryo chorioallantoic membrane assay. Both the H13A and H114A mutant proteins compete effectively with angiogenin in the latter assay; only a 5-fold molar excess of H13A over unmodified protein is required for complete inhibition. The His----Ala substitutions, however, do not have any significant effect on the interaction of angiogenin with human placental ribonuclease inhibitor, an extremely potent inhibitor of angiogenin (Ki approximately 7 x 10(-16 M) previously shown to interact with another active-site residue, Lys-40. The effects of more conservative replacements-glutamine at position 13 and asparagine at position 114--were also examined. While the enzymatic activity of the H114N mutant was at least 3300-fold less than for the unmodified protein, the H13Q derivative had only 300-fold reduced activity toward tRNA and cytidylyl(3'----5') adenosine. Both substitutions substantially decreased angiogenic activity. The parallel effects on ribonucleolytic and biological activities observed with all four mutant proteins provide strong evidence that the latter activity of angiogenin is dependent on a functional enzymatic active site. The capacity of the H13A and H114A derivatives to compete with angiogenin in the chorioallantoic membrane assay suggests several additional features of the biological mode of action of this protein.     

Role of H164 in a unique dye-decolorizing heme peroxidase DyP

The expression system of a unique dye-decolorizing peroxidase DyP in Escherichia coli has been constructed. The molecular mass of the expressed DyP (eDyP) is 47kDa, indicating no any modification with saccharides. The characteristics of eDyP were almost the same as those of native DyP from a fungus Thanatephorus cucumeris Dec 1 and recombinant DyP with Aspergillus oryzae except thermostability. As H164 was suggested to be the proximal histidine based on the preliminary X-ray crystallographic analysis of DyP, the site-directed mutations H164A and H166A (residue near H164) were introduced into the gene encoding DyP. The specific activity and RZ value of the purified H164A were 1.52U/mg and 0.11, respectively, which were 99.8% and 95% lower than those of eDyP, respectively. On the contrary, those of H166A were not different from those of eDyP. Therefore, H164 was confirmed to be the proximal histidine.     

Significance of metal ions in galactose-1-phosphate uridylyltransferase: an essential structural zinc and a nonessential structural iron.

Galactose-1-phosphate uridylyltransferase (GalT) catalyzes the reversible transformation of UDP-glucose and galactose-1-phosphate (Gal-1-P) into UDP-galactose and glucose-1-phosphate (Glc-1-P) by a double displacement mechanism, with the intermediate formation of a covalent uridylyl-enzyme (UMP-enzyme). GalT is a metalloenzyme containing 1.2 mol of zinc and 0.7 mol of iron/mol of subunits [Ruzicka, F. J., Wedekind, J. E., Kim, J., Rayment, I., and Frey, P. A. (1995) Biochemistry 34, 5610-5617]. The zinc site lies 8 A from His 166 in active site, and the iron site lies 30 A from the active site [Wedekind,J. E., Frey, P. A., & Rayment, I. (1995) Biochemistry 34, 11049-11061]. Zinc is coordinated in tetrahedral geometry by Cys 52, Cys 55, His 115, and His 164. His 164 is part of the highly conserved active-site triad His 164-Pro 165-His 166, in which His 166 is the nucleophilic catalyst. Iron is coordinated in square pyramidal geometry with His 296, His 298, and Glu 182 in bidentate coordination providing the base ligands and His 281 providing the axial ligand. In the present study, site-directed mutagenesis, kinetic, and metal analysis studies show that C52S-, C55S-, and H164N-GalT are 3000-, 600-, and 10000-fold less active than wild-type. None of the variants formed the UMP-enzyme in detectable amounts upon reaction with UDP-Glc in the absence of Gal-1-P. Their zinc content was very low, and the zinc + iron content was about 50% of that for wild-type GalT. Mutation of His 115 to Asn 115 resulted in decreased activity to 2.9% of wild-type, with retention of zinc and iron. In contrast to the zinc-binding site, Glu 182 in the iron site is not important for enzymatic activity. The variant E182A-GalT displayed about half the activity of wild-type GalT, and all of the active sites underwent uridylylation to the UMP-enzyme, similar to wild-type GalT, upon reaction with UDP-Glc. Metal analysis showed that while E182A-GalT contained 0.9 equiv of zinc/subunit, it contained no iron. The residual zinc can be removed by dialysis with 1,10-phenanthroline, with the loss in activity being proportional to the amount of residual zinc. It is concluded that the presence of zinc is essential for maintaining GalT function, whereas the presence of iron is not essential.     

Escherichia coli alkaline phosphatase: X-ray structural studies of a mutant enzyme (His-412-->Asn) at one of the catalytically important zinc binding sites.

The X-ray structure of a mutant version of Escherichia coli alkaline phosphatase (H412N) in which His-412 was replaced by Asn has been determined at both low (-Zn) and high (+Zn) concentrations of zinc. In the wild-type structure, His-412 is a direct ligand to one of the two catalytically critical zinc atoms (Zn1) in the active site. Characterization of the H412N enzyme in solution revealed that the mutant enzyme required high concentrations of zinc for maximal activity and for high substrate and phosphate affinity (Ma L, Kantrowitz ER, 1994, J Biol Chem 269:31614-31619). The H412N enzyme was also inhibited by Tris, in contrast to the wild-type enzyme, which is activated more than twofold by 1 M Tris. To understand these kinetic properties at the molecular level, the structure of the H412N (+Zn) enzyme was refined to an R-factor of 0.174 at 2.2 A resolution, and the structure of the H412N(-Zn) enzyme was refined to an R-factor of 0.166 at a resolution of 2.6 A. Both indicated that the Asn residue substituted for His-412 did not coordinate well to Zn1. In the H412N(-Zn) structure, the Zn1 site had very low occupancy and the phosphate was shifted by 1.8 A from its position in the wild-type structure. The Mg binding site was also affected by the substitution of Asn for His-412. Both structures of the H412N enzyme also revealed a surface-accessible cavity near the Zn1 site that may serve as a binding site for Tris.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure and mechanism of an ADP-glucose phosphorylase from Arabidopsis thaliana

The X-ray crystal structure of the At5g18200.1 protein has been determined to a nominal resolution of 2.30 A. The structure has a histidine triad (HIT)-like fold containing two distinct HIT-like motifs. The sequence of At5g18200.1 indicates a distant family relationship to the Escherichia coli galactose-1-P uridylyltransferase (GalT): the determined structure of the At5g18200.1 protein confirms this relationship. The At5g18200.1 protein does not demonstrate GalT activity but instead catalyzes adenylyl transfer in the reaction of ADP-glucose with various phosphates. The best acceptor among those evaluated is phosphate itself; thus, the At5g18200.1 enzyme appears to be an ADP-glucose phosphorylase. The enzyme catalyzes the exchange of (14)C between ADP-[(14)C]glucose and glucose-1-P in the absence of phosphate. The steady state kinetics of exchange follows the ping-pong bi-bi kinetic mechanism, with a k(cat) of 4.1 s(-)(1) and K(m) values of 1.4 and 83 microM for ADP-[(14)C]glucose and glucose-1-P, respectively, at pH 8.5 and 25 degrees C. The overall reaction of ADP-glucose with phosphate to produce ADP and glucose-1-P follows ping-pong bi-bi steady state kinetics, with a k(cat) of 2.7 s(-)(1) and K(m) values of 6.9 and 90 microM for ADP-glucose and phosphate, respectively, at pH 8.5 and 25 degrees C. The kinetics are consistent with a double-displacement mechanism that involves a covalent adenylyl-enzyme intermediate. The X-ray crystal structure of this intermediate was determined to 1.83 A resolution and shows the AMP group bonded to His(186). The value of K(eq) in the direction of ADP and glucose-1-P formation is 5.0 at pH 7.0 and 25 degrees C in the absence of a divalent metal ion, and it is 40 in the presence of 1 mM MgCl(2).     

A water-mediated salt link in the catalytic site of Escherichia coli alkaline phosphatase may influence activity

Escherichia coli alkaline phosphatase catalyzes the hydrolysis of a wide variety of phosphomonoesters at similar rates, and the reaction proceeds through a phosphoenzyme intermediate. The active site region is highly conserved between the E. coli and mammalian alkaline phosphatases. The three-dimensional structure of the E. coli enzyme indicates that Lys-328, which is replaced by histidine in all mammalian alkaline phosphatases, is bridged to the phosphate through a water molecule. This water molecule is also hydrogen bonded to Asp-327, a bidendate ligand of the one of the two zinc atoms. Here we report the use of site-specific mutagenesis to convert Lys-328 to both histidine and alanine. Steady-state kinetic studies above pH 7.0 indicate that both mutant enzymes have altered pH versus activity profiles compared to the profile for the wild-type enzyme. At pH 10.3, in the presence of Tris, the Lys-328----Ala enzyme is approximately 14-fold more active than the wild-type enzyme. At the same pH in the absence of Tris the Lys-328----Ala enzyme is still 6-fold more active than the wild-type enzyme. Both mutant enzymes have lower phosphate affinities than the wild-type enzyme at all pH values investigated. Pre-steady-state kinetics at pH 5.5 reveal that the Lys-328----Ala enzyme behaves very similar to the phosphate-free wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)