Essential Catalytic Residues Research Articles

Mutagenesis and characterization of specific residues in fatty acid ethyl ester synthase: a gene for alcohol-induced cardiomyopathy.

Fatty acid ethyl ester synthase-III metabolizes both ethanol and carcinogens. Structure-function studies of the enzyme have not been performed in relation to site specific mutagenesis. In this study, three residues (Gly 32, Cys 39 and His 72) have been mutated to observe their role in enzyme activity. Gly to Gln, Cys to Trp and His to Ser mutations did not affect fatty acid ethyl ester synthase activity, but His to Ser mutant had less than 9% of control glutathione S-transferase activity. The apparent loss of transferase activity reflected a 28 fold weaker binding constant for glutathione. Thus, this study indicates that Gly and Cys may not be important for synthase or transferase activities however, histidine may play a role in glutathione binding, but it is not an essential catalytic residue of glutathione S-transferase or for fatty acid ethyl ester synthase activity.

Mutational analysis of the three cysteines and active-site aspartic acid 103 of ketosteroid isomerase from Pseudomonas putida biotype B

In order to clarify the roles of three cysteines in ketosteroid isomerase (KSI) from Pseudomonas putida biotype B, each of the cysteine residues has been changed to a serine residue (C69S, C81S, and C97S) by site-directed mutagenesis. All cysteine mutations caused only a slight decrease in the k(cat) value, with no significant change of Km for the substrate. Even modification of the sulfhydryl group with 5,5'-dithiobis(2-nitrobenzoic acid) has almost no effect on enzyme activity. These results demonstrate that none of the cysteines in the KSI from P. putida is critical for catalytic activity, contrary to the previous identification of a cysteine in an active-site-directed photoinactivation study of KSI. Based on the three-dimensional structures of KSIs with and without dienolate intermediate analog equilenin, as determined by X-ray crystallography at high resolution, Asp-103 was found to be located within the range of the hydrogen bond to the equilenin. To assess the role of Asp-103 in catalysis, Asp-103 has been replaced with either asparagine (D103N) or alanine (D103A) by site-directed mutagenesis. For D103A mutant KSI there was a significant decrease in the k(cat) value: the k(cat) of the mutant was 85-fold lower than that of the wild-type enzyme; however, for the D103N mutant, which retained some hydrogen bonding capability, there was a minor decrease in the k(cat) value. These findings support the idea that aspartic acid 103 in the active site is an essential catalytic residue involved in catalysis by hydrogen bonding to the dienolate intermediate.

Cleavage specificity of a proteolytic antibody light chain and effects of the heavy chain variable domain

The recombinant light chain (L chain) of an antibody raised by immunization with vasoactive intestinal polypeptide (VIP) cleaved this peptide on the C-terminal side of basic residues. The major sites of cleavage in VIP were two adjacent peptide bonds, Lys20Lys21 and Lys21Tyr22. Lower levels of cleavage were evident at Arg14Lys15 and Lys15Gln16. Hydrolysis of radiolabeled VIP by the L chain was inhibited by two serine protease inhibitors, diisopropylfluorophosphate and aprotinin, but not by soybean or lima bean trypsin inhibitors or inhibitors of other classes of proteases. To probe the role of the VH domain, single chain Fv constructs composed of the VL domain of the anti-VIP L chain linked via a 14-residue peptide to its natural VH domain partner or an irrelevant anti-lysozyme VH domain (hybrid Fv) were prepared. The anti-VIP Fv hydrolyzed VIP with Ks 21.4-fold lower than the L chain and 250-fold lower than the hybrid Fv, suggesting increased affinity for the substrate ground state due to the anti-VIP VH domain. The kinetic efficiency (kcat/Ks) of the anti-VIP Fv was 6.6-fold greater compared to the L chain and 29.4-fold greater compared to the hybrid Fv. Peptide-MCA substrates unrelated in sequence to VIP were hydrolyzed by the anti-VIP Fv and L chain at equivalent rates. These observations lead to a model of catalysis by the anti-VIP Fv in which the essential catalytic residues are located in the VL domain and additional residues from the VH domain are involved in high affinity binding of the substrate.

Relationship of conserved residues in the IMP binding site to substrate recognition and catalysis in Escherichia coli adenylosuccinate synthetase.

Gln34, Gln224, Leu228, and Ser240 are conserved residues in the vicinity of bound IMP in the crystal structure of Escherichia coli adenylosuccinate synthetase. Directed mutations were carried out, and wild-type and mutant enzymes were purified to homogeneity. Circular dichroism spectroscopy indicated no difference in secondary structure between the mutants and the wild-type enzyme in the absence of substrates. Mutants L228A and S240A exhibited modest changes in their initial rate kinetics relative to the wild-type enzyme, suggesting that neither Leu228 nor Ser240 play essential roles in substrate binding or catalysis. The mutants Q224M and Q224E exhibited no significant change in KmGTP and KmASP and modest changes in KmIMP relative to the wild-type enzyme. However, kcat decreased 13-fold for the Q224M mutant and 10(4)-fold for the Q224E mutant relative to the wild-type enzyme. Furthermore, the Q224E mutant showed an optimum pH at 6.2, which is 1.5 pH units lower than that of the wild-type enzyme. Tryptophan emission fluorescence spectra of Q224M, Q224E, and wild-type proteins under denaturing conditions indicate comparable stabilities. Mutant Q34E exhibits a 60-fold decrease in kcat compared with that of the wild-type enzyme, which is attributed to the disruption of the Gln34 to Gln224 hydrogen bond observed in crystal structures. Presented here is a mechanism for the synthetase, whereby Gln224 works in concert with Asp13 to stabilize the 6-oxyanion of IMP.

L-allo-threonine aldolase from Aeromonas jandaei DK-39: gene cloning, nucleotide sequencing, and identification of the pyridoxal 5'-phosphate-binding lysine residue by site-directed mutagenesis.

We have isolated the gene encoding L-allo-threonine aldolase (L-allo-TA) from Aeromonas jandaei DK-39, a pyridoxal 5'-phosphate (PLP)-dependent enzyme that stereospecifically catalyzes the interconversion of L-allo-threonine and glycine. The gene contains an open reading frame consisting of 1,014 nucleotides corresponding to 338 amino acid residues. The protein molecular weight was estimated to be 36,294, which is in good agreement with the subunit molecular weight of the enzyme determined by polyacrylamide gel electrophoresis. The enzyme was overexpressed in recombinant Escherichia coli cells and purified to homogeneity by one hydrophobic column chromatography step. The predicted amino acid sequence showed no significant similarity to those of the currently known PLP-dependent enzymes but displayed 40 and 41% identity with those of the hypothetical GLY1 protein of Saccharomyces cerevisiae and the GLY1-like protein of Caenorhabditis elegans, respectively. Accordingly, L-allo-TA might represent a new type of PLP-dependent enzyme. To determine the PLP-binding site of the enzyme, all of the three conserved lysine residues of L-allo-TA were replaced by alanine by site-directed mutagenesis. The purified mutant enzymes, K51A and K224A, showed properties similar to those of the wild type, while the mutant enzyme K199A was catalytically inactive, with corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys199 of L-allo-TA probably functions as an essential catalytic residue forming an internal Schiff base with PLP of the enzyme to catalyze the reversible aldol reaction.

Development of "substrate-trapping" mutants to identify physiological substrates of protein tyrosine phosphatases.

The identification of substrates of protein tyrosine phosphatases (PTPs) is an essential step toward a complete understanding of the physiological function of members of this enzyme family. PTPs are defined by a conserved catalytic domain harboring 27 invariant residues. From a mutagenesis study of these invariant residues that was guided by our knowledge of the crystal structure of PTP1B, we have discovered a mutation of the invariant catalytic acid (Asp-181 in PTP1B) that converts an extremely active enzyme into a "substrate trap." Expression of this D181A mutant of PTP1B in COS and 293 cells results in an enzyme that competes with endogenous PTP1B for substrates and promotes the accumulation of phosphotyrosine primarily on the epidermal growth factor (EGF) receptor as well as on proteins of 120, 80, and 70 kDa. The association between the D181A mutant of PTP1B and these substrates was sufficiently stable to allow isolation of the complex by immunoprecipitation. As predicted for an interaction between the substrate-binding site of PTP1B and its substrates, the complex is disrupted by vanadate and, for the EGF receptor, the interaction absolutely requires receptor autophosphorylation. Furthermore, from immunofluorescence studies, the D181A mutant of PTP1B appeared to retain the endogenous EGF receptor in an intracellular complex. These results suggest that the EGF receptor is a bona fide substrate for PTP1B in vivo and that one important function of PTP1B is to prevent the inappropriate, ligand-independent, activation of newly synthesized EGF receptor in the endoplasmic reticulum. This essential catalytic aspartate residue is present in all PTPs and has structurally equivalent counterparts in the dual-specificity phosphatases and the low molecular weight PTPs. Therefore we anticipate that this method may be widely applicable to facilitate the identification of substrates of other members of this enzyme family.

Importance of the gamma-carboxyl group of glutamate-462 of the large alpha-subunit for the catalytic function and the stability of the multienzyme complex of fatty acid oxidation from Escherichia coli.

His450 of the large alpha-subunit of the multienzyme complex of fatty acid oxidation from Escherichia coli was recently identified as an essential catalytic residue of L-3-hydroxyacyl-CoA dehydrogenase [He, X-Y., & Yang, S.-Y. (1996) Biochemistry 35, 9625-9630]. To explore the roles of acidic residues in the dehydrogenase catalysis, every conserved acidic residue in the dehydrogenase functional domain except for those in the NAD-binding motif was replaced with alanine. The resulting mutant complexes were overproduced and characterized. Their component enzymes other than the dehydrogenase were affected very slightly. Removal of the beta-carboxyl group of Asp524 and Asp542 caused only a 3- and 4-fold, respectively, decrease in the catalytic efficiency of the dehydrogenase, thereby showing that their involvement in the dehydrogenase catalysis was limited. In contrast, the alpha/Glu462-->Ala mutant complex showed a greater than 160-fold reduction in the kcat of the dehydrogenase in the forward direction without a significant change of the k(m) for the substrate. The catalytic properties of the alpha/Glu462-->Gln mutant complex were found to be similar to those of the alpha/Glu462-->Ala mutant complex except that the kcat of the dehydrogenase in the backward direction was about 4-fold lower and the Km for the substrate of the thiolase was 6-fold higher. It is concluded that the negative charge of the gamma-carboxyl group of Glu462, but not its ability to form a hydrogen bond, is critical for its interaction with His450, thereby assisting in the catalysis of the dehydrogenase. The pKa of His450 in the E.NADH binary complex was virtually unchanged by the replacement of Glu462 with Ala or Gln. It seems that the binding of substrate is necessary for forming a strong interaction between His450 and Glu462 with the result that the electroneutrality in the active site is maintained and the activation energy of the reaction is lowered. Additionally, the negative charge of Glu462 increases the thermostability of the multienzyme complex.

Proteolytic Antibodies

AbstractCatalytic activities are known to arise naturally in antibodies. Several naturally‐occurring peptides, synthetic protease substrates, DNA, and esters are known to be cleaved by antibodies. There is increased production of antigen‐specific catalytic antibodies in autoimmune disease. Polyreactive catalytic antibodies are present in unimmunized donors. Antibody light chains isolated from multiple myeloma patients frequently express proteolytic activity. Immunization protocols using as immunogens the ground state of a naturally‐occurring polypeptide, anti‐enzyme antibodies, or the enzyme itself are known to provoke catalytic antibody synthesis. Active site residues in the light chain subunit serve as the catalytic residues in a model antibody with peptide bond cleaving activity. A split‐site model in which distinct amino acids serve as the essential catalytic residues and substrate ground‐state recognition residues has been developed from mutagenesis studies. Engineering of the available antibodies could potentially generate improved catalysts. The possible mechanisms underlying proteolysis by natural antibodies and evolution of the catalytic activity are reviewed.

Steroid Recognition by Chloramphenicol Acetyltransferase: Engineering and Structural Analysis of a High Affinity Fusidic Acid Binding Site

The antibiotic fusidic acid and certain closely related steroidal compounds are potent competitive inhibitors of the type I variant of chloramphenicol acetyltransferase (CAT I). In the absence of crystallographic data for CAT I, the structural determinants of steroid binding were identified by (1) construction in vitroof genes encoding chimaeric enzymes containing segments of CAT Iand the related type III variant (CAT III) and (2) site-directed mutagenesis of the gene encoding CAT III, followed by kinetic characterisation of the substituted variants. Replacement of four residues of CAT III(Gln92, Asn146, Tyr168 and Ile172) by their equivalents from CAT Iyields an enzyme variant that is susceptible to competitive inhibition by fusidate with respect to chloramphenicol ( K i=5.4 μM). The structure of the complex of fusidate and the Q92C/N146F/Y168F/I172V variant, determined at 2.2 Å resolution by X-ray crystallography, reveals the inhibitor bound deep within the chloramphenicol binding site and in close proximity to the side-chain of His195, an essential catalytic residue. The aromatic side-chain of Phe146 provides a critical hydrophobic surface which interacts with non-polar substituents of the steroid. The remaining three substitutions act in concert both to maintain the appropriate orientation of Phe 146 and viaadditional interactions with the bound inhibitor. The substitution of Gln92 by Cys eliminates a critical hydrogen bond interaction which constrains a surface loop (residues 137 to 142) of wild-type CAT IIIwhich must move in order for fusidate to bind to the enzyme. Only two hydrogen bonds are observed in the CAT-fusidate complex, involving the 3-α-hydroxyl of the A-ring and both hydroxyl of Tyr25 and NE2 of His195, both of which are also involved in hydrogen bonds with substrate in the CAT III-chloramphenicol complex. In the acetyl transfer reaction catalysed by CAT, NE2, of His195 serves as a general base in the abstraction of a proton from the 3-hydroxyl of chloramphenicol as the first chemical step in catalysis. The structure of the CAT-inhibitor complex suggests that deprotonation of the 3-α-hydroxyl of bound fusidate by this mechanism could produce an oxyanion nucleophile analogous to that seen with chloramphenicol, but one which is incorrectly positioned to attack the thioester carbonyl of acetyl-CoA, accounting for the observed failure of CAT to acetylate fusidate.

Site-directed Mutagenic Alteration of Potential Active-site Residues of the A Subunit of Escherichia coli Heat-labile Enterotoxin: EVIDENCE FOR A CATALYTIC ROLE FOR GLUTAMIC ACID 112

Escherichia coli heat-labile enterotoxin (LT) and the related cholera toxin exert their effects on eukaryotic cells through the ADP-ribosylation of guanine nucleotide-binding proteins of the adenylate cyclase complex. The availability of the crystal structure for LT has permitted the tentative identification of residues that lie within or are vicinal to a presumptive NAD(+)-binding site and thus may play a role in substrate binding or catalysis. Using a plasmid clone encoding the A subunit of LT, we have introduced substitutions at such potential active-site residues and analyzed the enzymatic properties of the resultant mutant analogs. Enzymatic analyses, employing both transducin and agmatine as acceptor substrates, revealed that substitutions at serine 61, glutamic acid 110, and glutamic acid 112 resulted in reduction of enzyme activity to < 10% of wild-type levels. Kinetic analyses indicated that alteration of these sites affected the catalytic rate of the enzyme and had little or no effect on the binding of either NAD+ or agmatine. Of the mutant analogs analyzed, only glutamic acid 112 appeared to represent an essential catalytic residue as judged by the relative effects on kcat and kcat/Km. The results provide formal evidence that glutamic acid 112 of the A subunit of LT represents a functional homolog or analog of catalytic glutamic acid residues that have been identified in several other bacterial ADP-ribosylating toxins and that it may play an essential role in rendering NAD+ susceptible to nucleophilic attack by an incoming acceptor substrate.

Characterization of a Dominant Negative Mutant of the Cell Cycle Ubiquitin-conjugating Enzyme Cdc34

The yeast Saccharomyces cerevisiae CDC34 gene encodes a ubiquitin-conjugating enzyme that is required for the cell cycle G1/S transition. We show here that a dominant negative Cdc34 protein is generated by simultaneously replacing both Cys95 and Leu99 with Ser residues. Cys95 is an essential catalytic residue that forms a transient thiol ester with ubiquitin during catalysis, and Leu99 is highly conserved among all known ubiquitin-conjugating enzymes. Mutants that encode either an alanine or a serine at one or both of these two positions are inactive. Of these eight mutants, overexpression of CDC34-C95S,L99S in wild type strains was found to block cell growth. Although cells overexpressing Cdc34-C95S,L99S do not exhibit the characteristic multibudded phenotype of cdc34 temperature-sensitive or null mutants, this blockade is relieved by simultaneous overxpression of wild type Cdc34. Purified Cdc34-C95S,L99S protein can be shown to inhibit in vitro ubiquitination of the Cdc34-specific substrate, Cln2 protein. We suggest that Cdc34-C95S,L99S selectively sequesters a subset of Cdc34 substrates or regulators. These findings have implications for the structure/function relationships of ubiquitin-conjugating enzymes, and suggest a general method for identifying components and substrates of specific ubiquitination pathways of eukaryotes.

Structure-reactivity relationships for beta-galactosidase (Escherichia coli, lac Z). 1. Brønsted parameters for cleavage of alkyl beta-D-galactopyranosides.

Seven substituted alkyl beta-D-galactopyranosides 1-OR have been prepared and shown to be fair to excellent substrates for hydrolysis catalyzed by beta-galactosidase (Escherichia coli, lac Z). Brønsted parameters of (beta 1g)k3 = -0.49 +/- 0.13 and (beta 1g)kcat/Km = -0.75 +/- 0.14, respectively, were determined at pH 8.6 for k3 (s-1), the first-order rate constant for cleavage of enzyme-bound 1-OR, and kcat/Km (M-1 s-1), the second-order rate constant for reaction of the free enzyme and 1-OR. There is a weak correlation between log Km and the pKa of the alkyl alcohol leaving group, which is attributed to stabilization of the Michaelis complex by hydrophobic interactions between the enzyme and electron-withdrawing halogen substituents at the alkoxy leaving group. These binding interactions are probably both productive and expressed in the value of kcat/Km and nonproductive and expressed in the value of k3. The negative values of beta 1g are inconsistent with enzymatic catalysis of endocyclic cleavage of the glycosidic bond. The values of beta 1g for enzyme-catalyzed cleavage of alkyl beta-D-galactopyranosides lie between those observed for the spontaneous (beta 1g approximately -1.25) and specific-acid-catalyzed (beta 1g approximately 0) cleavage of acetals, and these pathways are therefore excluded for the enzyme-catalyzed reaction. Removal of the metal cofactor Mg2+ from the enzyme causes a approximately 0.2 unit decrease in (beta 1g)k3 for beta-galactosidase-catalyzed cleavage of 1-OR. The interpretation of this change in beta 1g is unclear. The Brønsted coefficients for the beta-galactosidase-catalyzed reaction are consistent with participation by an essential catalytic residue in concerted general-acid catalysis of cleavage of the glycosidic bond of 1-OR and/or stabilization of developing negative charge at the alkoxy oxygen by interaction with the magnesium ion cofactor.

Aminolevulinate synthase: lysine 313 is not essential for binding the pyridoxal phosphate cofactor but is essential for catalysis.

5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in animals and some bacteria. Lysine-313 of the mouse erythroid aminolevulinate synthase was recently identified to be linked covalently to the pyridoxal 5'-phosphate cofactor (Ferreira GC, Neame PJ, Dailey HA, 1993, Protein Sci 2:1959-1965). Here we report on the effect of replacement of aminolevulinate synthase lysine-313 by alanine, histidine, and glycine, using site-directed mutagenesis. Mutant enzymes were purified to homogeneity, and the purification yields were similar to those of the wild-type enzyme. Although their absorption spectra indicate that the mutant enzymes bind pyridoxal 5'-phosphate, they bind noncovalently. However, addition of glycine to the mutant enzymes led to the formation of external aldimines. The formation of an external aldimine between the pyridoxal 5'-phosphate cofactor and the glycine substrate is the first step in the mechanism of the aminolevulinate synthase-catalyzed reaction. In contrast, lysine-313 is an essential catalytic residue, because the K313-directed mutant enzymes have no measurable activity. In summary, site-directed mutagenesis of the aminolevulinate synthase active-site lysine-313, to alanine (K313A), histidine (K313H), or glycine (K313G) yields enzymes that bind the pyridoxal 5'-phosphate cofactor and the glycine substrate to produce external aldimines, but which are inactive. This suggests that lysine-313 has a functional role in catalysis.

Expression inEscherichia coliof a Gene Encoding a Thermostable Chitinase fromStreptomyces thermoviolaceusOPC-520

An expression plasmid for a thermostable chitinase gene from S. thermoviolaceus OPC-520 in E. coli was constructed. A cloned chitinase (Chi40) was purified from the periplasmic space of E. coli harboring the expression plasmid. The N-terminal sequence of Chi40 was 11 amino acids longer than that of chitinase from S. thermovilaceus OPC-520 (ST chitinase), however, a loss or addition of the amino acid residues did not affect the enzymatic properties. The mutations of Asp-145 and Glu-147 drastically decreased the specific activity of chitinase from the wild type, indicating that both amino acid residues are the best candidates for the essential catalytic residues of Chi40.

Functional Analysis of Conserved Histidines in Choline Acetyltransferase by Site‐Directed Mutagenesis

The choline acetyltransferase (ChAT) reaction involves the transfer of the acetyl group of acetyl-CoA to choline, in which an active site histidine is believed to act as a general acid/base catalyst. A comparison of the deduced amino acid sequences of the enzyme from Drosophila, pig, rat, and Caernohabditis elegans revealed three conserved histidines: Drosophila His268, His393, and His426. Each of these histidines was replaced by a leucine and a glutamine, and the kinetic properties of each of the recombinant mutant enzymes were determined. The mutations yielded active His268Leu-ChAT, His268Gln-ChAT, and His393Gln-ChAT and inactive His393Leu-ChAT, His426Leu-ChAT, and His426Gln-ChAT. The kinetic constants Km(CoA), Km(acetylcholine), and Vmax were essentially the same for all of the active mutants. When the integrity of the CoASAc binding site was investigated in the inactive mutants, the data suggested that the binding site in His393Leu-ChAT is disrupted but conserved in His426Leu-ChAT and His426Gln-ChAT. These results suggest that His426 is an essential catalytic residue and could serve as an acid/base catalyst.

Lysine 87 in the beta subunit of tryptophan synthase that forms an internal aldimine with pyridoxal phosphate serves critical roles in transimination, catalysis, and product release.

This study provides valuable insights into the functions of the lysine residue that forms an internal aldimine with pyridoxal phosphate in the beta subunit of tryptophan synthase from Salmonella typhimurium. Our spectroscopic and kinetic studies demonstrate that a mutant alpha 2 beta 2 complex having beta subunit lysine 87 replaced by threonine forms external aldimines with several amino acids including L-serine, beta-chloro-1-alanine, L-tryptophan, and D-tryptophan. Because the rates of aldimine formation are very slow, we conclude that one role of lysine 87 in the wild type enzyme is to facilitate formation of external aldimines by transimination. Lysine 87 is an essential catalytic residue because the mutant alpha 2 beta 2 complex has no measurable activity in reactions catalyzed by the beta subunit and does not convert external aldimines to products. The mutant enzyme carries out two slow partial beta-elimination reactions: the conversion of beta-chloro-L-alanine and L-serine to enzyme-bound aminoacrylate. The reaction with L-serine is catalyzed by ammonia, which partially replaces the deleted epsilon-amino group. Lysine 87 is important for substrate and product release because L-serine, L-tryptophan, and aminoacrylate dissociate very slowly from the mutant alpha 2 beta 2 complex. Our ability to prepare very stable derivatives of the mutant alpha 2 beta 2 complex containing tightly bound aldimines with a substrate, a product, or a reaction intermediate provides valuable materials for ongoing x-ray crystallographic investigations and future kinetic analyses of the allosteric activation of the alpha subunit by beta subunit ligands.

Characterization and sequencing of rabbit, pig and mouse angiogenins: discernment of functionally important residues and regions

Rabbit, pig and mouse angiogenins have been purified from blood serum and characterized, and the rabbit and pig proteins have been sequenced fully. A partial sequence of the mouse protein is consistent with the sequence deduced from the genomic DNA (Bond, M.D. and Vallee, B.L. (1990) Biochem. Biophys. Res. Commun. 171, 988–995). All three angiogenins are homologous to the pancreatic RNases and contain the essential catalytic residues His-13, Lys-40 and His-114, and the 6 half-cystines of the human protein. Like human angiogenin they display extremely low ribonucleolytic activities toward wheat-germ RNA, yeast RNA, poly(C) and poly(U). The rabbit and pig proteins induce neovascularization in vivo and also inhibit protein synthesis in vitro. The interaction of rabbit, pig and bovine angiogenins with placental ribonuclease inhibitor, a potent inhibitor of angiogenin, was examined by fluorescence spectroscopy. Rate and equilibrium binding constants indicate that rabbit angiogenin binds to the inhibitor much like human angiogenin, whereas the pig and bovine proteins show significant differences. A comparison of the five angiogenin sequences now available points to specific residues that are highly conserved among them but differ from the corresponding residues in the RNases. These residues are clustered in particular regions of the three-dimensional structure, two of which contribute to the angiogenic, second-messenger and/or protein synthesis inhibition activities of human angiogenin.

X-ray structures of two single-residue mutants of DNase I: H134Q and Y76A.

The structures of the single-residue mutants H134Q and Y76A of bovine pancreatic DNase I have been determined and refined including data to 2.3 and 2.4 A resolution respectively, by X-ray crystallography. H134 is an essential catalytic residue, while Y76 contributes to the binding of DNA by providing a large van der Waals contact area that stabilizes the wide minor groove seen in DNase I-DNA complexes. The mutant proteins, which show strongly reduced activities of 0.001% (H134Q) and 0.3% (Y76A), were expressed in E. coli and both crystallize in space-group C2 with almost identical unit cells. The crystal packing scheme is different from that found in wild type crystals grown under very similar conditions, presumably due to the absence of the carbohydrate moiety. In both mutants the conformation of the protein is nearly identical to that of the wild type enzyme and changes are confined to surface loops involved in packing. The disruption of the hydrogen bonds between H134, E78 and Y76 in both mutants leads to an increased mobility and positional shifts in the DNA-binding loop, mainly around residue Y76. This in turn may further reduce DNA-binding affinity and, thus, contribute to the low activity. In contrast, symmetry contacts involving residues 97-108 lead to a stabilization of the flexible loop compared to wild type DNase I.

Site-directed mutagenesis of the active site cysteine in Klebsiella aerogenes urease.

Cysteine 319 in the large subunit of Klebsiella aerogenes urease was identified as an essential catalytic residue based on chemical modification studies (Todd, M.J., and Hausinger, R.P. (1991) J. Biol. Chem. 266, 24327-24331). Through site-directed mutagenesis, this cysteine has been changed independently to alanine, serine, aspartate, and tyrosine. None of these mutations (C319A, C319S, C319D, and C319Y, respectively) affected the size or level of synthesis of the urease subunits as monitored by polyacrylamide gel electrophoresis. The wild type enzyme and each of the mutant proteins was purified and their properties were compared. The C319Y protein possessed no detectable activity, while activity was reduced in C319A, C319S, and C319D to 48, 4.5, and 0.03% of wild type levels under normal assay conditions. All of the active mutants had a small increase in Km when compared to the wild type value. The active mutants displayed a greatly reduced sensitivity to inactivation by iodoacetamide in comparison to the wild type enzyme, confirming our previous assignment of the essential cysteine to this residue based on active site peptide mapping. In contrast to the wild type enzyme, inactivation of the mutant proteins was not affected by the presence of the competitive inhibitor phosphate, suggesting that the remaining slow rate of iodoacetamide inactivation is due to modification away from the active site. The pH dependence of urease activity was substantially altered in the active mutants with C319S and C319D showing a pH optimum near 5.2, and C319A near 6.7, compared to the pH 7.75 optimum of wild type urease. These data are consistent with Cys-319 facilitating catalysis at neutral and basic pH values by participating as a general acid.

Role of threonine 190 in modulating the catalytic function of malate dehydrogenase from a thermophile Thermus flavus

Random mutagenesis of malate dehydrogenase from a thermophilic bacterium, Thermus flavus AT-62, had revealed that a Thr190----Ile replacement near the essential catalytic residue His187 caused marked modulation of the catalytic properties. For further exploration of a role of the residue at this position, this residue was substituted with each of the other amino acids by site-directed mutagenesis. Most of the mutations except for substitution with Ser caused increases in Km for oxaloacetate and increases Ki for oxaloacetate of 2-110 times. Substitution with His or Pro was characterized by the complete loss of substrate inhibition, along with a marked increase in Km for oxaloacetate. Kinetic analyses of the native and altered malate dehydrogenases at various pHs revealed that both Km and Ki for oxaloacetate decreased proportionally to the decrease in pH from 8.40 to 5.75, whereas kcat was nearly constant within the pH range. Apparent shifts of the optimum pH values toward acidity observed with most of the altered malate dehydrogenases were attributed to the increase in Ki, which facilitated the release from the substrate inhibition at a lower pH. Replacement of Thr310, a possible counterpart with which Thr190 forms a hydrogen bond, by Ile caused changes in the catalytic properties similar to those of the Thr190-substituted enzymes. These results suggest that not only the loss of the hydrogen bond between Thr190 and Thr310 but also properties of the residues introduced at position 190 cause modulation of the catalytic properties, probably through dislocation of the loop structure that contains the catalytic residue His187.