Phospholipase A of sea snake Laticauda semifasciata venom. Isolation and properties of novel forms lacking tryptophan.

The venom gland extracts of the sea snake Laticauda semifasciata contained at least four forms of phospholipase A separable on a CM-cellulose column. They were designated as phospholipases A I-IV in the order of elution from the column. Phospholipases A I, III, and IV were isolated in a homogeneous state. They were similar to one another in amino acid composition and molecular weight (14,000) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phospholipase A I contained one tryptophan residue. whereas III and IV did not. Although all these forms had the same A2-type positional specificity, they were classified into two groups (I, and III and IV) on the basis of enzymic properties. Phospholipase A I had a higher specific activity and showed normal kinetics, whereas III and IV had approximately one-tenth of the specific activity of I and showed biphasic kinetics due to their activation by the reaction products. Phospholipase A I, the major form, seems to be identical with phospholipase A reported previously (Tu, A.T., Passey, R.B., & Toom, P.M. (1970) Arch. Biochem. Biophys. 140, 96-106), whereas the other two, III and IV, are new. Phospholipase A I became more like III and IV in enzymic properties on modification with N-bromosuccinimide.     

Function of conserved aromatic residues in the Gal/GalNAc-glycosyltransferase motif of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1

UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc transferases), which initiate mucin-type O-glycan biosynthesis, have broad acceptor substrate specificities, and it is still unclear how they recognize peptides with different sequences. To increase our understanding of the catalytic mechanism of GalNAc-T1, one of the most ubiquitous isozymes, we studied the effect of substituting six conserved aromatic residues in the highly conserved Gal/GalNAc-glycosyltransferase motif with leucine on the catalytic properties of the enzyme. Our results indicate that substitutions of Trp302 and Phe325 have little impact on enzyme function and that substitutions of Phe303 and Tyr309 could be made with only limited impact on the interaction(s) with donor and/or acceptor substrates. By contrast, Trp328 and Trp316 are essential residues for enzyme functions, as substitution with leucine, at either site, led to complete inactivation of the enzymes. The roles of these tryptophan residues were further analyzed by evaluating the impact of substitutions with additional amino acids. All evaluated substitutions at Trp328 resulted in enzymes that were completely inactive, suggesting that the invariant Trp328 is essential for enzymatic activity. Trp316 mutant enzymes with nonaromatic replacements were again completely inactive, whereas two mutant enzymes containing a different aromatic amino acid, at position 316, showed low catalytic activity. Somewhat surprisingly, a kinetic analysis revealed that these two amino acid substitutions had a moderate impact on the enzyme's affinity for the donor substrate. By contrast, the drastically reduced affinity of the Trp316 mutant enzymes for the acceptor substrates suggests that Trp316 is important for this interaction.     

Effect of alkylation of tryptophan residues on the enzymatic and pharmacological properties of snake venom phospholipase A2

Snake venom phospholipases A2 show a remarkable degree of amino acid sequence homology yet differ markedly in enzymatic and pharmacological activities. The basic phospholipase A2 from Naja nigricollis venom has much greater lethal potency, cardiotoxicity, hemolytic and anticoagulant activity than the acidic or neutral enzymes from Naja naja atra or Hemachatus haemachatus venoms, respectively, even though it has lower enzymatic activity than the latter two enzymes. Previous studies in which we selectively modified lysine and free carboxyl groups suggested that the pharmacological and enzymatic active sites are not identical. Tryptophan residues have been suggested as being involved in substrate binding although some phospholipases have no tryptophan. We investigated the effect of alkylating the tryptophans in N. nigricollis, N. n. atra, and H. haemachatus phospholipases A2 with 2-hydroxy-5-nitrobenzyl bromide. Chemical modification caused decreases in enzymatic activity, although the extent of inactivation varied with the enzyme and with the substrate (lecithin micelles, egg yolk, heart homogenates). The specificity of the enzymes for individual phospholipid substrates was not affected. Alkylation of the tryptophans also caused decreases in lethal, hemolytic, anticoagulant, and cardiotoxic potencies, which were similar to the extents of decrease in enzymatic activity. Our results suggest that tryptophans are not specifically associated with either the enzymatic or the pharmacological active site nor are essential for either activity.     

Ammodytoxin A, a highly lethal phospholipase A2 from Vipera ammodytes ammodytes venom

The amino acid sequence of ammodytoxin A, the most toxic presynaptically active phospholipase A2 isolated from Vipera ammodytes ammodytes venom, was determined. The primary structure was deduced from peptides obtained by Staphylococcus aureus proteinase and trypsin digestion of reduced and carboxymethylated protein and from the automated Edman degradation of the N-terminal part of the non-reduced molecule. According to the sequence, the enzyme classifies to the subgroup IIA of the phospholipase A2 family of enzymes. The location of basic residues believed to be responsible for the toxic activity of presynaptically active phospholipases differs substantially from those in the highly toxic enzymes of other subgroups. Comparison of the sequence with sequences of other snake venom enzymes indicates that the toxic site(s) may not be the same in all subgroups of presynaptically active phospholipases.     

The primary structure of rat platelet phospholipase A2

In our previous report (Hayakawa, M., Kudo, I., Tomita, M., & Inoue, K. (1988) J. Biochem. 103, 263-266), we have shown that phospholipases A2 purified from rat platelet membrane fractions and an extracellular medium of thrombin-stimulated rat platelets were essentially identical to each other. Both purified enzymes were digested with proteases, and the resulting peptides were subjected to primary sequence determination. The sequence analysis of the HPLC-separated peptides and the alignment of the sequences showed a tentative primary structure of rat platelet phospholipase A2, which was composed of 125 amino acid residues. It showed 47% homology with snake venom Agkistrodon halys blomhoffii phospholipase A2.     

Role of the invariant Asn345 and Asn435 residues in a leucine aminopeptidase from Bacillus kaustophilus as evaluated by site-directed mutagenesis

Role of the conserved Asn345 and Asn435 residues of Bacillus kaustophilus leucine aminopeptidase (BkLAP) was investigated by performing computer modeling and site-directed mutagenesis. Replacement of BkLAP Asn345 with Gln or Leu resulted in a dramatic reduction in enzymatic activity. A complete loss of the LAP activity was observed in Asn435 variants. Circular dichroism spectra were nearly identical for wild-type and all mutant enzymes, while measurement of intrinsic tryptophan fluorescence revealed the significant alterations of the microenvironment of aromatic amino acid residues in Asn345 and Asn435 replacements. Except for N435R and N435L, wild-type and other mutant enzymes showed a similar sensitivity towards temperature-induced denaturation. Computer modeling of the active-site structures of wild-type and mutant enzymes exhibits a partial or complete loss of the hydrogen bonding in the variants.     

The Evolution of Acetyl-CoA Synthase

Acetyl-coenzyme A synthases (ACS) are Ni-Fe-S containing enzymes found in archaea and bacteria. They are divisible into 4 classes. Class I ACS's catalyze the synthesis of acetyl-CoA from CO2 + 2e-, CoA, and a methyl group, and contain 5 types of subunits (alpha, beta, gamma, delta, and epsilon). Class II enzymes catalyze essentially the reverse reaction and have similar subunit composition. Class III ACS's catalyze the same reaction as Class I enzymes, but use pyruvate as a source of CO2 and 2e-, and are composed of 2 autonomous proteins, an alpha 2 beta 2 tetramer and a gamma delta heterodimer. Class IV enzymes catabolize CO to CO2 and are alpha-subunit monomers. Phylogenetic analyses were performed on all five subunits. ACS alpha sequences divided into 2 major groups, including Class I/II sequences and Class III/IV-like sequences. Conserved residues that may function as ligands to the B- and C-clusters were identified. Other residues exclusively conserved in Class I/II sequences may be ligands to additional metal centers in Class I and II enzymes. ACS beta sequences also separated into two groups, but they were less divergent than the alpha's, and the separation was not as distinct. Class III-like beta sequences contained approximately 300 residues at their N-termini absent in Class I/II sequences. Conserved residues identified in beta sequences may function as ligands to active site residues used for acetyl-CoA synthesis. ACS gamma-sequences separated into 3 groups (Classes I, II, and III), while delta-sequences separated into 2 groups (Class I/II and III). These groups are less divergent than those of alpha sequences. ACS epsilon-sequence topology showed greater divergence and less consistency vis-à-vis the other subunits, possibly reflecting reduced evolutionary constraints due to the absence of metal centers. The alpha subunit phylogeny may best reflect the functional diversity of ACS enzymes. Scenarios of how ACS and ACS-containing organisms may have evolved are discussed.     

Biphasic kinetics of the colchicine-tubulin interaction: role of amino acids surrounding the A ring of bound colchicine molecule

Isotypes of vertebrate tubulin have variable amino acid sequences, which are clustered at their C-terminal ends. Isotypes bind colchicine at different on-rates and affinity constants. The kinetics of colchicine binding to purified (unfractionated) brain tubulin have been reported to be biphasic under pseudo-first-order conditions. Experiments with individual isotypes established that the presence of beta(III) in the purified tubulin is responsible for the biphasic kinetics. Because the isotypes mainly differ at the C termini, the colchicine-binding kinetics of unfractionated tubulin and the beta(III) isotype, cleaved at the C termini, have been tested under pseudo-first-order conditions. Removal of the C termini made no difference to the nature of the kinetics. Sequence alignment of different beta isotypes of tubulin showed that besides the C-terminal region, there are differences in the main body as well. To establish whether these differences lie at the colchicine-binding site or not, homology modeling of all beta-tubulin isotypes was done. We found that the isotypes differed from each other in the amino acids located near the A ring of colchicine at the colchicine-binding site on beta tubulin. While the beta(III) isotype has two hydrophilic residues (serine(242) and threonine(317)), both beta(II) and beta(IV) have two hydrophobic residues (leucine(242) and alanine(317)). beta(II) has isoleucine at position 318, while beta(III) and beta(IV) have valine at that position. Thus, these alterations in the nature of the amino acids surrounding the colchicine site could be responsible for the different colchicine-binding kinetics of the different isotypes of tubulin.     

The amino acid sequence of toxin IV from the Androctonus australis scorpion: differing effects of natural mutations in scorpion alpha-toxins on their antigenic and toxic properties.

The complete amino acid sequence (64 residues) of the AaH IV toxin from the scorpion Androctonus australis Hector was determined by automated Edman degradation and was compared with the sequences of other Androctonus toxins. AaH IV was also tested by radioimmunoassay for binding to antisera raised against other toxins of the same species. The results indicated that AaH IV shares some of the antigenic properties of AaH I and AaH III toxins, but does not cross-react with anti-AaH II antibodies. The structural basis for the observed antigenic relationships can be found in the high degree of homology displayed by AaH IV with regard to AaH I and III, the changes in amino acid residues equally affecting regions included or excluded from the main predicted antigenic sites of AaH IV. The lower biological potency of AaH IV is presumably the result of some of the sequence differences. In particular, substitution affecting the charge and bulkiness of residue 61 could account for the poor receptor binding and consequential weak toxic properties of this molecule.     

Purification, partial characterization, and immunological relationships of multiple low molecular weight protease inhibitors of soybean.

Five protease inhibitors, I--V, in the molecular weight range 7000--8000 were purified from Tracy soybeans by ammonium sulfate precipitation, gel filtration on Sephadex G-100 and G-75, and column chromatography on DEAE-cellulose. In common with previously described trypsin inhibitors from legumes, I--V have a high content of half-cystine and lack tryptophan. By contrast with other legume inhibitors, inhibitor II contains 3 methionine residues. Isoelectric points range from 6.2 to 4.2 in order from inhibitor I to V. Molar ratios (inhibitor/enzyme) for 50% trypsin inhibition are I = 4.76, II = 1.32, III = 3.22, IV = 2.17, V = 0.97. Only V inhibit chymotrypsin significantly (molar ratio = 1.33 for 50% inhibition). The sequence of the first 16 N-terminal amino acid residued of inhibitor V is identical to that of the Bowman-Birk inhibitor; all other observations also indicate that inhibitor V and Bowman-Birk are identical. The first 20 N-terminal amino acid residues of inhibitor II show high homology to those of Bowman-Birk inhibitor, differing by 1 deletion and 5 substitutions. Immunological tests show that inhibitors I through IV are fully cross-reactive with each other but are distinct from inhibitor V.     

Purification and Properties of the Trypsin Inhibitors from Buckwheat Seed

The trypsin inhibitors in buckwheat seeds were isolated by affinity chromatography on trypsin-Sepharose 4B, and the components were fractionated by chromatography on DEAE-Sepharose CL-6B. The major components, inhibitors I, II and III, were found to be homogeneous proteins with molecular weight of about 8,000. Trypsin inhibitory activity was more pronounced than the chymotrypsin inhibitory activity in all the inhibitor preparation obtained. The three major inhibitors had similar amino acid compositions and had no detectable amounts of tryptophan and carbohydrate. A high level of acidic and basic amino acid residues and a low level of methionine, tyrosine and phenylalanine residues characterized the inhibitors. Although the inhibitors I and II were particularly thermostable, inhibitor III, the most abundant component, was shown to be relatively heat-labile.     

Structure-function relationships of phospholipases. The anticoagulant region of phospholipases A2

In an effort to identify the anticoagulant region of venom phospholipases A2, we have systematically compared the amino acid sequences of strong, weak and non-anticoagulant phospholipases. The comparison disclosed several significant substitutions in the region between residues 54 and 77 (homology numbers). This proposed anticoagulant region is positively charged in strong, but negatively charged in weak and non-anticoagulant phospholipases. The microenvironment of a tryptophan residue falls within the proposed region, accounting for the differential characteristics of intrinsic fluorescence changes observed at 335 nm after the binding of phospholipid vesicles to strong and weak anticoagulants. Four lysine residues are located in specific positions in the "anticoagulant" region of strong anticoagulants, and should form a cationic surface, based on analogy with the available crystallographic structures. The chemical modification of lysine, arginine, tyrosine, and tryptophan residues and carboxylate groups, performed by other investigators, not only provides added support for the predicted site, but also confirms the essentiality of the positive charges in the site. This region may participate in the formation of a specific preferential hydrolytic complex leading to the strong anticoagulant effect. The anticoagulant region is distinct and separate from the predicted neurotoxic and myotoxic sites, and is located on the opposite surface of the phospholipase molecule.     

Proteinase Inhibitors in Plant Seeds

The proteinase inhibitors I (R-I) and III (R-III) isolated from Japanese radish seed were characterized in terms of their N-terminal amino acids, amino acid composition and reacting groups. The amino acid composition of two proteins differed from each other, while histidine, methionine and tryptophan contents were all low. N-Terminal amino acids of these inhibitors determined by Edman degradation were the same; valine.

By modifying free amino groups in the inhibitors with trinitrobenzenesulfonic acid, R-III was greatly inactivated in proportion to the modification of amino groups, but the activity of R-I was not affected.

However, modification of arginyl residues of R-I by cyclohexanedione reduced its activity. These results indicate that R-I is an arginine-type and R-III is a lysine-type inhibitor.     

Amino acid sequences of two ferredoxins from pokeweed, Phytolacca americana

The amino acid sequences of two ferredoxins isolated from pokeweed, Phytolacca americana, were determined. Tryptic peptides of maleyl-carboxymethyl-ferredoxin I and carboxymethyl-ferredoxin II were prepared and analyzed. The large peptides were further digested with staphylococcal protease and chymotrypsin. Ferredoxins I and II were composed of 96 and 98 amino acid residues, respectively. Though ferredoxin I lacks tryptophan and methionine, ferredoxin II contains both of them. In a comparison of the amino acid sequences with those of other higher plant ferredoxins, ferredoxin I is one residue shorter than others at the carboxyl-terminus and ferredoxin II one longer than others at the amino-terminus. Ferredoxins I and II differ in 23 sites from each other and in 27 to 37 sites from other higher plant ferredoxins. This suggests that duplication of the ferredoxin gene occurred after the divergence of pokeweed from other higher plants. A phylogenetic tree including all other ferredoxins was constructed.     

Mechanism of DNA binding by the DnaB helicase of Escherichia coli: analysis of the roles of domain gamma in DNA binding.

We have analyzed the mechanism of single-stranded DNA (ssDNA) binding mediated by the C-terminal domain gamma of the DnaB helicase of Escherichia coli. Sequence analysis of this domain indicated a specific basic region, "RSRARR", and a leucine zipper motif that are likely involved in ssDNA binding. We have carried out deletion as well as in vitro mutagenesis of specific amino acid residues in this region in order to determine their function(s) in DNA binding. The functions of the RSRARR domain in DNA binding were analyzed by site-directed mutagenesis. DnaBMut1, with mutations R(328)A and R(329)A, had a significant decrease in the DNA dependence of ATPase activity and lost its DNA helicase activity completely, indicating the important roles of these residues in DNA binding and helicase activities. DnaBMut2, with mutations R(324)A and R(326)A, had significantly attenuated DNA binding as well as DNA-dependent ATPase and DNA helicase activities, indicating that these residues also play a role in DNA binding and helicase activities. The role(s) of the leucine zipper dimerization motif was (were) determined by deletion analysis. The DnaB Delta 1 mutant with a 55 amino acid C-terminal deletion, which left the leucine zipper and basic DNA binding regions intact, retained DNA binding as well as DNA helicase activities. However, the DnaB Delta 2 mutant with a 113 amino acid C-terminal deletion that included the leucine zipper dimerization motif, but not the RSRARR sequence, lost DNA binding, DNA helicase activities, and hexamer formation. The major findings of this study are (i) the leucine zipper dimerization domain, I(361)-L(389), is absolutely required for (a) dimerization and (b) ssDNA binding; (ii) the base-rich RSRARR sequence is required for DNA binding; (iii) three regions of domain gamma (gamma I, gamma II, and gamma III) differentially regulate the ATPase activity; (iv) there are likely three ssDNA binding sites per hexamer; and (v) a working model of DNA unwinding by the DnaB hexamer is proposed.     

Functional residues on the enzyme active site of glyoxalase I from bovine brain

Bovine brain glyoxalase I was investigated in order to identify amino acid residues essential for its catalytic activity. This enzyme is a 44-kDa dimeric protein which exhibits a characteristic intrinsic fluorescence, with an emission peak centered at 342 nm. The total of eight tryptophan residues/molecule was estimated by using a fluorescence titration method. Low values of Stern Volmer quenching constants for the quenchers used indicated that the tryptophan residues are relatively buried in the native molecule. Similar results were obtained for glyoxalase I, purified from yeast and human erythrocytes. The activity of bovine brain glyoxalase I was found to be particularly sensitive to 2,3-butanedione and diethylpyrocarbonate, selective reagents for arginine and histidine residues, respectively. A minor effect was observed by treatment of the enzyme with other amino acid-specific reagents. A protective effect of the competitive inhibitor S-hexylglutathione was observed for all reagents used, indicating the presence of modified amino acids in or near the enzyme active site.     

Purification, some properties of two phospholipases A2 (CM-I and CM-II) and the amino-acid sequence of CM-II from Aspidelaps scutatus (shield or shield-nose) venom

Two phospholipases A2, CM-I and CM-II, from Aspidelaps scutatus venom were purified by gel filtration followed by ion-exchange chromatography on CM-cellulose. The enzymes consist of 119 amino acids including fourteen half-cystines. The complete primary structure of CM-II has been determined. The sequence and the invariant amino acid residues resemble those of the phospholipase A2 from the genus Naja. The toxicity of the enzymes is comparable to those encountered for the phospholipases A2 from African cobra venoms. The phospholipase A2 (CM-II) contains two histidine residues which are located at position 20 and the reactive site (histidine-47) of the enzyme.