Negative ion postsource decay time-of-flight mass spectrometry of peptides containing acidic amino acid residues

Acidic peptides have been studied by negative ion postsource decay (PSD) matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The peptides contained from 5 to 16 residues and were chosen on the basis of their patterns of the acidic residues. Using typical MALDI sample preparation techniques employing an acidic matrix, gastrin I (1-14), and epidermal mitosis inhibiting pentapeptide yielded much larger deprotonated ion signals, [M - H]-, than protonated ions, [M + H]+. This may be due to their absence of basic residues, coupled with their arrays of acidic residues. The PSD fragmentation of the peptide negative ions showed that an array of acidic residues, as in gastrin I (1-14), yielded simple spectra containing mainly backbone cleavage ions from the C-terminus. Hirudin (54-65), which contains two sets of two consecutive Glu residues, and fibrinopeptide A and fibrinopeptide B, with isolated acidic residues, also showed backbone cleavages as common fragment ions. In addition, the two sets of isolated consecutive amino acid residues in Cys(Bzl)84-CD4 (81-92) and hirudin (54-56) yielded internal ions from the cleavages at the (O=C)-NH bond between the acidic residues. Also observed were ions with unique side chain losses, such as the loss of C6H4O from a tyrosine residue and SCH2C6H5 and CH2C6H5 from a benzylated cysteine residue. Compared to the positive mode, the negative-ion PSD yielded fewer fragments which usually involved only one type of backbone cleavage (e.g., [Yn - H2O]-). These simple spectra aided interpretation. Overall, the acidic peptides studied yielded negative ion PSD spectra that were useful for peptide sequencing.     

Proteases in renal cell death: calpains mediate cell death produced by diverse toxicants

In recent years, the combination of gel electrophoresis and mass spectrometry has developed into one of the most powerful approaches for the analysis of proteins. However, a number of gel electrophoresis-induced protein modifications have been described. Cysteine is the most endangered amino acid readily reacting with mercaptoethanol or free acrylamide. In the course of studies on glucan phosphorylases (E.C.2.4.1.1) from white potato (Solanum tuberosum L.) and the T cell receptor, we noticed that proteolytic peptides from these proteins can undergo an unexpected modification, giving rise to a mass increment of 14 Da. By post-source decay (PSD) analysis the modification was identified as methylation of the glutamic acid side chain carboxyl group. The methylation takes place during Coomassie blue staining of proteins if both trichloroacetic acid and methanol are present in the staining solution. Replacement of methanol by ethanol under otherwise unchanged conditions results in ethylation of the peptides. The in vitro alkylation was further studied by using synthetic peptides which contain, at different positions: glutamic acid, aspartic acid or the corresponding amides. The kinetic analysis of the observed reactions revealed that glutamic acid is preferentially methylated. The three other amino acid residues can be methylated but with a velocity at least one order of magnitude lower. Although these modifications complicate the interpretation of the spectra, they provide valuable structural information.     

Modification of aspartic acid residues to induce trypsin cleavage

1,2-Diaminoethane and diaminomethane were coupled to aspartic acid residues in small peptides by means of a water-soluble carbodiimide. The resulting modified side chains sufficiently resembled lysine for trypsin to cleave the peptides. Similar modification of glutamic acid residues in peptides gave little or no susceptibility to trypsin.     

Direct sequencing of neuropeptides in biological tissue by MALDI-PSD mass spectrometry

Dissected tissue pieces of the pituitary pars intermedia from the amphibian Xenopus laevis was directly subjected to matrix-assisted laser desorption/ionization (MALDI) mass analysis. The obtained MALDI peptide profile revealed both previously known and unexpected processing products of the proopiomelanocortin gene. Mass spectrometric peptide sequencing of a few of these neuropeptides was performed by employing MALDI combined with postsource decay (PSD) fragment ion mass analysis. The potential of MALDI-PSD for sequence analysis of peptides directly from unfractionated tissue samples was examined for the first time for the known desacetyl-alpha-MSH-NH2 and the presumed vasotocin neuropeptide. In addition, the sequence of an unknown peptide which was present in the pars intermedia tissue sample at mass 1392.7 u was determined. The MALDI-PSD mass spectrum of precursor ion 1392.7 u contained sufficient structural information to uniquely identify the sequence by searching protein sequence databases. The determined amino acid sequence corresponds to the vasotocin peptide with a C-terminal extension of Gly-Lys-Arg ("vasotocinyl-GKR"), indicating incomplete processing of the vasotocin precursor protein in the pituitary pars intermediate of X. laevis. Both vasotocin and vasotocinyl-GKR are nonlinear peptides containing a disulfide (S-S) bridge between two cysteine residues. Interpretation of the spectra of these two peptides reveals three different forms of characteristic fragment ions of the cysteine side chain: peptide-CH2-SH (regular mass of Cys-containing fragment ions), peptide-CH2-S-SH (regular mass + 32 u) and peptide = CH2 (regular mass -34 u) due to cleavage on either side of the sulfur atoms.     

Amino acid alterations in the benA (beta-tubulin) gene of Aspergillus nidulans that confer benomyl resistance

We report the cloning and sequencing of 18 mutant alleles of the benA, beta-tubulin gene of Aspergillus nidulans that confer resistance to the benzimidazole antifungal, antimicrotubule compounds benomyl, carbendazim, nocodazole, and thiabendazole. In 12 cases, amino acid 6 was changed from histidine to tyrosine or leucine. In four cases, amino acid 198 was changed from glutamic acid to aspartic acid, glutamine, or lysine. In two cases, amino acid 200 was altered from phenylalanine to tyrosine. These data, along with previous data indicating that amino acid 165 is involved in the binding of the R2 group of these compounds [Jung and Oakley, 1990: Cell Motil. Cytoskeleton 17:87-94], suggest that regions of beta-tubulin containing amino acids 6, 165, and 198-200 interact to form the binding site of benzimidazole antimicrotubule agents. These results also suggest that the presence of phenylalanine at amino acid 200 contributes to the great sensitivity of many fungi to benzimidazole antimicrotubule agents.     

Characterization of pathotype-specific epitopes of newcastle disease virus fusion glycoproteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and post-source decay sequencing

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) was used to characterize the F2 polypeptide of the fusion (F) protein of an avirulent isolate (VRI 82-6409) of Newcastle disease virus (NDV) that was previously identified by immunochemical screening as having a variant cleavage activation sequence in its fusion protein precursor (F0). The major glycoform of the intact F2 polypeptide of the VRI 82-6409 isolate was 89 Da smaller than the F2 polypeptide of the avirulent V4 isolate of the Queensland strain of NDV. Analysis of AspN protease digests of the F2 polypeptides by MALDI/TOF-MS, with and without high-performance liquid chromatographic (HPLC) separation, showed this mass difference to be due to a combination of differences in the extents of glycosylation and an amino acid difference in the AspN peptides derived from the C-termini of the F2 polypeptides. Accuracies achieved in analysis of the AspN peptides allowed the identification of this amino acid difference as glutamic acid in the VRI 82-6409 isolate compared with glycine in the V4 isolate. Analysis of fragments formed by post-source decay (PSD) of ions of the C-terminal AspN peptides localized the difference to the C-terminal residues of the respective F2 polypeptides. The present study demonstrated that MALDI/TOF-MS is a highly effective technique for the characterization of NDV variants identified by immunochemical screening of pathotype-specific epitopes at the C-termini of their F2 polypeptides.     

Determination of the hydroxyapatite-nucleating region of bone sialoprotein

Bone sialoprotein (BSP) was shown to be a potent nucleator of hydroxyapatite (HA) in a steady-state agarose gel system (Hunter and Goldberg, 1993, PNAS 90: 8562). Nucleation of HA was also demonstrated with the homopolymer poly-glutamic acid but not with poly-aspartic acid or osteopontin. Since BSP contains contiguous sequences of glutamic acid, it is reasonable to suggest that the HA-nucleating activity of BSP resides within these regions. Purified porcine BSP was treated with trypsin and digests fractionated by gel filtration. In addition to small peptides (P3-5), two peptides of 38 kDa (P1) and 25 kDA (P2) were recovered, and after characterization assigned to the regions within BSP encompassing residues 133-272 (P1) and 42-125 (P2). Each of these peptides contained one of the two glutamic acid-rich regions of porcine BSP. In the steady-state agarose gel system, BSP, P1 and P2 induced HA formation, whereas the pooled small BSP-derived peptides (P3-5) did not. Analysis by circular dichroism spectroscopy revealed that the homopolymer poly-L-glutamic acid assumes a helical structure, while poly-L-aspartic acid does not. These findings suggest that the nucleating activity does not require intact molecules, that the nucleation of HA and BSP appears to require glutamic acid-rich sequences in a helical conformation and that there are two domains in porcine BSP that are each capable of nucleating HA.     

Prompt fragmentation of disulfide-linked peptides during matrix-assisted laser desorption ionization mass spectrometry

During the analysis of an Asp-N digest of a recombinant hematopoietic growth factor by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), we observed pseudomolecular ions corresponding to reduced forms of peptides known to be present only in single disulfide linkages. Chromatographic fractionation of the peptide digest, followed by MALDI-MS and electrospray ionization (ESI) MS, confirmed that the reduced peptides were not present in the map. Fragmentation of the disulfide-linked peptides into their reduced forms occurred upon ionization from different matrices (alpha-cyano-4-hydroxycinnamic acid,2,5-dihydroxybenzoic acid, and in some instances sinapinic acid) but only after increasing the laser fluence to above threshold. Analysis of the disulfide-linked peptide fractions by ESI-MS, before and after mixing and drying with matrix, indicated that the matrix did not cause reduction. In a low-energy tandem mass spectrometric experiment with one of the cystinyl peptides, fragmentation did not occur preferentially at the disulfide bond. The pseudomolecular ions exhibited the same m/z values by MALDI-MS as their chemically reduced counterparts, indicating that they arose due to prompt fragmentation or "in-source decay" rather than "post-source decay". This finding is important for MALDI-MS analysis of peptide maps of proteins and peptide fractions with intact disulfides.     

Separation and identification of peptides in single neurons by microcolumn liquid chromatography-matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and postsource decay analysis

Microcolumn liquid chromatography (LC) was interfaced with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for separation and identification of peptides present in single neurons from the brain of the snail Lymnaea stagnalis. The nanoliter microcolumn LC effluent, mixed off-line with nanoliter matrix solution, was deposited onto the sample target every 60 s, producing fractions of approximately 145 nL in volume, which, upon drying, produced spots of approximately 1 mm in size. At the end of the chromatographic separation, fractions from the sample target were scanned by MALDI-TOF-MS. Identification of peptide peaks was achieved on the basis of LC elution order and mass information. Further identification based on sequence information was carried out for a native peptide fractionated by microcolumn LC from a single neuron with the postsource decay technique.     

Improving mass spectrometric sequencing of arginine-containing peptides by derivatization with acetylacetone

Modification of arginine residues in bradykinin, [1-5]-bradykinin, splenopentin and two synthetic pentapeptides with acetylacetone (pentane-2,4-dione) significantly increases the relative abundance of sequence-specific fragment ions produced by matrix-assisted laser desorption/ionization (MALDI). The fragmentation efficiency as measured by post-source decay in a reflectron time-of-flight mass spectrometer increases by a factor of 2-3.5. Peptide bonds adjacent to modified residues are more susceptible to cleavage than in the non-derivatized peptide ions. The increased lability of these bonds gives rise to more complete sequence information. In addition, the relative abundances of sequence-specific fragment ions are enhanced. This strategy makes it possible to obtain valuable structural information from arginine-containing peptides that otherwise do not fragment well.     

The yeast mic2 mutant is defective in the formation of mannosyl-diinositolphosphorylceramide

Three transmembrane glutamic acid residues play essential roles in the metal-tetracycline/H+ antiporter Tet(K) of Staphylococcus aureus [Fujihira et al., FEBS Lett. 391 (1996) 243-246]. In the putative hydrophilic loop region of the Tet(K) and Tet(L) proteins, six acidic residues are conserved. Asp74, Asp200, Asp318 and Glu381 are located on the putative cytoplasmic side, and Asp39 and Glu345 on the putative periplasmic side. These residues were replaced by a neutral amino acid residue or a charge-conserved one. In contrast to the transmembrane glutamic acid residues, the replacement of the two glutamic acid residues (Glu345 and Glu381) did not affect the tetracycline resistance level. Out of the other four aspartic acid residues, the only essential residue is Asp318, any replacement of which resulted in complete loss of the tetracycline resistance and transport activity. Asp318 is located in cytoplasmic loop 10-11 in the putative 14-transmembrane-segment topology of Tet(K). In the case of the tetracycline exporters of Gram-negative bacteria, the only essential acidic residue in the cytoplasmic loop region is located in loop 2-3 [Yamaguchi et al., Biochemistry 31 (1992) 8344-8348]. It may be a general role for tetracycline efflux proteins that three transmembrane and one cytoplasmic acidic residues are mandatory for the tetracycline transport function.     

Identification of catalytic bases in the active site of Escherichia coli methylglyoxal synthase: cloning, expression, and functional characterization of conserved aspartic acid residues

Methylglyoxal synthase provides bacteria with an alternative to triosephosphate isomerase for metabolizing dihydroxyacetone phosphate (DHAP). In the present studies, the methylglyoxal synthase gene in Escherichia coli has been cloned and sequenced. The identified open reading frame (ORF) codes for a polypeptide of 152 amino acids, consistent with the 17 kDa purified protein. The sequence of this protein is not similar to any other protein of known function, including the functionally similar protein triosephosphate isomerase. The methylglyoxal synthase gene was amplified by PCR, subcloned into the pET16B expression vector, and expressed in the host E. coli BL21(DE3). Sequence comparison of the methylglyoxal protein and related ORFs from four different bacterial species revealed that four aspartic acid and no glutamic acid residues are absolutely conserved. The function of the four aspartic acid residues was tested by mutating them to either asparagine or glutamic acid. Thermal denaturation, CD spectroscopy, and gel filtration experiments showed that the mutant enzymes had the same secondary and quaternary structure as the wild-type enzyme. Kinetic characterization of both Asp 71 and Asp 101 mutant proteins shows reduced kcat/Km by 10(3)- and 10(4)-fold respectively, suggesting that they are both intimately involved in catalysis. A time-dependent inhibition of both Asp 20 and Asp 91 asparagine mutants by DHAP suggests that these two residues are involved with protecting the enzyme from DHAP or reactive intermediates along the catalytic pathway. In combination with the results of 2-phosphoglycolate binding studies, a catalytic mechanism is proposed.     

The amino acid sequences of carboxypeptidases I and II from Aspergillus niger and their stability in the presence of divalent cations

The amino acid sequences of serine carboxypeptidase I (CPD-I) and II (CPD-II), respectively, from Aspergillus niger have been determined by conventional Edman degradation of the reduced and vinylpyridinated enzymes and peptides hereof generated by cleavage with cyanogen bromide, iodobenzoic acid, glutamic acid cleaving enzyme, AspN-endoproteinase and EndoLysC proteinase. CPD-I consists of a single peptide chain of 471 amino acid residues, three disulfide bridges and nine N-glycosylated asparaginyl residues, while CPD-II consists of a single peptide chain of 481 amino acid residues, has three disulfide bridges, one free cysteinyl residue and nine glycosylated asparaginyl residues. The enzymes are closely related to carboxypeptidase S3 from Penicillium janthinellum. Both Ca2+ and Mg2+ stabilize CPD-I as well as CPD-II, at basic pH values, Ca2+ being most effective, while the divalent ions have no effect on the activity of the two enzymes.     

Undifferentiated (embryonal) sarcoma of the liver: pathologic basis of imaging findings in 28 cases

An acidic 1,2-alpha-mannosidase from fungus, Aspergillus saitoi (now designated Aspergillus phoenicis), is highly specific for 1,2-alpha-mannosidic linkage in the high-mannose type oligosaccharide at pH 5.0. The predicted amino acid sequence of several peptide regions, including aspartic acid and glutamic acid, bears striking similarities to 1,2-alpha-mannosidases from fungi, yeast and mouse. Active site determination of the enzyme expressed in Saccharomyces cerevisiae cells was performed by site-directed mutagenesis. Substitutions of Asp-269 to Glu and of the Glu-residues, Glu-273, Glu-411, Glu-414 and Glu-474, to Asp altered the drastic decrease of specific activities with Man alpha 1-2Man-OMe and Man9-GlcNAc2-PA as substrates and shifted the optimal pH of the mutant enzymes. From the present results, Asp-269 is probably in the ionized COO- form, whereas one of four glutamic acid residues, probably Glu-411, is the un-ionized COOH form according to the analogy of a plausible mechanism for lysozyme catalysis. It is assumed that three glutamic acid residues, Glu-273, Glu-414, and Glu-474, are probably binding sites of substrate.     

Use of high-precision gas isotope ratio mass spectrometry to determine natural abundance 13C in lutein isolated from C3 and C4 plant sources

Previous studies have shown that cholesterol esterification activity by lecithin:cholesterol acyltransferase (LCAT) is progressively inhibited as up to three acidic acid residues are chemically modified. The purpose of this study was to determine whether three glutamic acid residues in LCAT (154, 155, and 165), that align exactly with three acidic acid residues (270, 271, and 281) in the amphipathic phospholipid binding region of apoE, were necessary for enzymatic activity. Site-directed mutagenesis was used to generate mutant constructs of LCAT in which glutamic acid residues 154, 155, and 165 were replaced with glutamine or lysine. Media harvested from transiently transfected COS cells was used as a source of LCAT for cholesterol esterification and phospholipase A2 (PLA2) assays. Cholesterol esterification for all mutant constructs (11-26 nmol CE/h/microg) was similar to or greater than that of wild type LCAT (16 nmol CE/h/microg), except for a triple mutant, in which glutamic acid residues 154, 155, and 165 were changed to lysines (5 nmol CE/h/microg). PLA2 activity followed a similar trend. There was a significant decrease in the cholesterol esterification to PLA2 activity ratio when residue 165 was mutated from its wild type negative charge (E) to an uncharged (Q) or positive (K) charged residue (10.2 vs. 6.0 vs. 4.3, respectively). We conclude that glutamic acid residues 154, 155, and 165 individually or collectively are not necessary for LCAT activity and that residue 165 may be in a region of LCAT that is involved with cholesterol binding or is sensitive to cholesterol binding at the active site of the enzyme.     

The importance of dominant negative effects of amino acid side chain substitution in peptide-MHC molecule interactions and T cell recognition

Previous studies on the role of specific residues of the peptide or MHC molecule in Ag presentation have revealed the sensitivity of this complex system to even small changes in structure. In our study, we have analyzed the effect of amino acid substitution in a major CD4+ T cell determinant (T1) of HIV-1 gp160 on binding and recognition in the context of various E alpha E beta MHC class II molecules. Individual alanine substitutions at all but three positions had little or no negative effect on either MHC binding or recognition by a specific T hybridoma, whereas substitutions with larger side chains often diminished reactivity. A poly-alanine peptide containing only four of the original residues was an effective MHC class II binder and in vivo immunogen, although lacking the ability to stimulate the hybridoma. Replacement of a glutamic acid in T1 with alanine or a size-conservative, uncharged glutamine, but not a negatively charged aspartic acid produced a peptide at least 100-fold more potent than the parent peptide, indicating an inhibitory effect of the negative charge. Conversely, substitution of a glutamic acid for valine at position 29 in the floor of the peptide binding site of the E alpha E beta molecule decreased functional presentation of this peptide by more than 2 logs. However, these two effects of glutamic acid were not complementary and were mediated by distinct mechanisms, as the change in the peptide altered the extent of binding to class II, but the change in the MHC molecule decreased recognition without inhibiting peptide binding. Taken together, the data all suggest the conclusion that changes in side-chains of peptides and MHC molecules affect Ag presentation and T cell stimulation most often by introducing dominant negative or interfering groups that prevent or alter the pattern of binding events primarily mediated by a very limited number of other residues in the Ag or presenting molecule. These results have important implications for understanding the biochemistry of peptide-MHC-TCR interactions and for the possible design of vaccines both more potent and less subject to allele-specific limitations on immunogenicity.     

Sequential attachment of arginine residues to peptides, catalyzed by subtilisin. 1. Effect of organic solvent composition

Subtilisin 72 sorbed on a macroporous glass catalyzed the condensation of Dnp(or Z)-Ala2-Leu-OCH3 with arginine amide in a mixture of DMSO and acetonitrile at a water content less than 0.07% (v/v). This reaction resulted in the sequential formation of peptides containing from one to four C-terminal arginine residues. The number of attached Arg residues depended on the DMSO concentration in the solvent mixture, which determined the local arginine excess on the sorbent surface, which significantly exceeded the molar arginine excess in the solution. This enzymic reaction opened up new opportunities for preparation of peptides with different content of arginine residues.     

Antigen mimicry in autoimmune disease sharing of amino acid residues critical for pathogenic T cell activation

A nonamer peptide from murine nicotinic acetylcholine receptor delta chain (ACR delta), which shared four amino acid residues with a nonamer peptide of murine ovarian zona pellucida glycoprotein ZP3, induced murine autoimmune oophoritis and IgG autoantibody to the zona pellucida. Crossreaction between the ACR delta and ZP3 peptides was established by the response of a ZP3 peptide-specific, oophoritogenic T cell clone to both peptides in association with IA (alpha k beta b). By substituting the ZP3 peptides with a single alanine, four amino acids within the ZP3 peptide were found to be important for ovarian autoimmune disease, autoantibody response, and stimulation of the ZP3-specific T cell clone. Substitution with conservative amino acids of three residues also ablated activity, whereas the fourth, a phenylalanine, was replaceable by tyrosine without loss of activity. Of the four critical amino acids, three were shared between the ZP3 peptide and the ACR delta peptide. Moreover, polyalanine peptides with the four critical ZP3 amino acids or the four amino acids common to the ZP3 and ACR delta peptides induced immune response to ZP3 and elicited severe ovarian autoimmune disease. Thus, organ-specific autoimmune disease can occur through immune response against unrelated self (or foreign) peptides that share with a self-peptide sufficient common amino acid residues critical for activation of pathogenic, autoreactive T cells.     

The amino acid sequence of topi pancreatic ribonuclease

Pancreatic tissue from topi (Damaliscus korrigum) contains three ribonuclease components in a ratio of 8:22:70. Two components are glycosidated, whereas the third one does not contain carbohydrate. The amino acid sequence of topi ribonuclease A was deduced from a tryptic digest of the performic acid-oxidized protein. Peptides were positioned by homology with other bovid ribonucleases. Only peptides that differed in amino acid composition from the corresponding peptides of bovine ribonuclease were sequenced. The evidence obtained for the sequence of residues 67-73 is incomplete. Among the bovid ribonucleases (cow, bison, eland, sheep, goat and gnu), topi ribonuclease shows the closest resemblance with sheep and goat ribonucleases; except that the glutamic acid residue at position 103 in the ribonucleases from sheep and goat is substituted by a lysine residue in topi. Topi ribonucleases A and B differ only in the presence of carbohydrate attached to asparagine 34.     

Characterization of pKa values and titration shifts in the cytotoxic ribonuclease alpha-sarcin by NMR. Relationship between electrostatic interactions, structure, and catalytic function

The electrostatic behavior of titrating groups in alpha-sarcin was investigated using 1H NMR spectroscopy. A total of 209 chemical shift titration curves corresponding to different protons in the molecule were determined over the pH range of 3.0-8.5. Nonlinear least-squares fits of the data to simple relationships derived from the Henderson-Hasselbalch equation led to the unambiguous determination of pKa values for all glutamic acid and histidine residues, as well as for the C-terminal carboxylate and most of the aspartic acids in the free enzyme. The ionization constants of catalytically relevant histidines, His50 and His137, and glutamic acid, Glu96, in the alpha-sarcin-2'-GMP complex were also determined. The pKa values of 15 ionizable groups (C-carboxylate, six aspartic acids, four glutamic acids, and four histidines) were found to be close to their normal values. On the other hand, a number of side chain groups, including those in the active center, showed pKa values far from their intrinsic values. Thus, the pKa values for active site residues His50, Glu96, and His137 were 7.7, 5.2, and 5.8 in the free enzyme and 7.6, approximately 4.8, and 6.8 in the alpha-sarcin-2'-GMP complex, respectively. The pKa values and the activity profile against ApA, as a function of pH, are in agreement with the proposed enzymatic mechanism (in common with RNase T1 and the family of the microbial ribonucleases), in which Glu96 and His137 act as a general base and general acid, respectively. In almost all microbial ribonucleases, a Phe-His interaction is present, which affects the pKa of one of the His residues at the active site (His137). The absence of this interaction in alpha-sarcin would explain the lower pKa value of this His residue, and provides an explanation for the decreased RNase activity of this protein as compared to those of other microbial ribonucleases.