Site specificity of casein kinase-2 (TS) from rat liver cytosol. A study with model peptide substrates

The factors determining the site recognition and phosphorylation by rat liver casein kinase-2 (CK-2) have been explored with a set of 14 related hexapeptides each including a single phosphorylatable amino acid and five acidic plus neutral residues. Such peptides are different from each other in the following features: the nature of the phosphorylatable amino acid, if any; its position relative to the critically required acidic residues; the extension and the structure of the acidic cluster. All of them were tested as substrate and/or competitive inhibitors of CK-2, and their kinetic and inhibition constants were determined. The results suggest the following conclusions. Under strictly comparable conditions Ser is by far preferred over Thr. Tyr not being affected at all. In order to carry out its role of structural determinant the critical acidic cluster must be located on the C-terminal side of the target residue, though not necessarily adjacent to it. The affinity for the protein-binding site, as deduced from Km and/or Ki values, is largely dependent on the number of acidic residues but it is also significantly enhanced if a hydroxylic residue is located on their N-terminal side. An acidic residue at position +3 relative to serine plays an especially important role for triggering phosphorylation, the peptide Ser-Glu-Glu-Ala-Glu-Glu having similar Km but negligible Vmax compared to Ser-Glu-Ala-Glu-Glu-Glu and Ser-Glu-Glu-Glu-Ala-Glu. These data provide a rationale for the substrate specificity of CK-2 and will give a helpful insight into the structure of the protein-binding site of this enzyme.     

Substrate-specificity determinants for a membrane-bound casein kinase of lactating mammary gland. A study with synthetic peptides

A tissue-specific casein kinase, purified from the Golgi-enriched-membrane fraction of guinea-pig lactating mammary gland (GEF-CK), readily phosphorylates the synthetic peptide Ser-Glu5, a good substrate of casein kinase-2, and several derivatives varying for the number and position of acidic residues on the C-terminal side of serine, except those lacking an acidic side chain at position +2. The least acidic peptide, still significantly affected by GEF-CK, is Ser-Ala-Glu-Ala3 which is not a substrate for CK-2. Conversely, the peptides Ser-Ala2-Glu-Ala2, Ser-Ala2-Glu3, Ser-Ala2-Glu5 and Ser-Glu-Ala-Glu3, all of which are more or less readily phosphorylated by CK-2, are not appreciably affected by GEF-CK. On the other hand the presence of additional glutamyl residues, besides the one in the second position, improves the affinity of the peptide substrate for GEF-CK, as indicated by the Km values of Ser-Glu5, Ser-Glu2-Ala3 and Ser-Ala-Glu-Ala3 which are 80, 950 and 3950 microM respectively. It is concluded that although both CK-2 and GEF-CK require, for optimal activity, rather extended acidic clusters on the C-terminal side of the target serine, the most critical residue in the case of GEF-CK is not the one at position +3, which is required for CK-2 catalyzed phosphorylation [Marin, O. et al. (1986) Eur. J. Biochem. 160, 239-244], but the one lying at position +2. Additional differences, concerning the site specificities of these enzymes, have been outlined using the threonyl derivative of Ser-Glu5 and the peptide Arg-Ser-Glu3-Val-Glu. The former is still phosphorylated by CK-2 but not to any appreciable extent by GEF-CK, which apparently is strictly specific for seryl residues. On the contrary, the presence of an N-terminal basic residue, which greatly reduces phosphorylation by CK-2, is tolerated rather well by GEF-CK. On the other hand a C-terminal basic residue, interrupting the acidic cluster, compromises phosphorylation by GEF-CK, as indicated by the extremely high Km value of Ser-Glu3-Lys-Glu vs Ser-Glu3-Val-Glu (13,000 and 170 microM, respectively).     

An analysis of the substrate specificity of insulin-stimulated protein kinase-1, a mammalian homologue of S6 kinase-II

The specificity determinants for insulin-stimulated protein kinase-I (ISPK-1) have been investigated with synthetic peptides based on naturally-occurring protein phosphoacceptor sequences. Peptides (Arg-Arg-Xaa-Ser-Xaa) that fulfill the consensus sequence for cyclic-AMP-dependent protein kinase (PK-A) are also phosphorylated readily by ISPK-1. The phosphorylation efficiency is improved by increasing the number of N-terminal arginine residues and by moving the arginyl cluster one residue further away from the serine, the nonapeptide (Arg)₄-Ala-Ala-Ser-Val-Ala being the best substrate among all the short peptides tested (K_m = 15 μM). Conversely, the substitution of either Thr for Ser or Lys for Arg is detrimental. Likewise, two flanking Pro residues and an Arg immediately N-terminal to the Ser act as negative specificity determinants. While the specificity of ISPK-1 shows several similarities to that of PK-A, including an absolute requirement for basic residues on the N-terminal side of the target Ser, it differs in several other respects including (1), the detrimental effect of a Lys for Arg substitution which is still compatible with some phosphorylation by ISPK-1, but not PK-A; (2), the presence of C-terminal acidic residues which are tolerated very well by ISPK-1, but are detrimental to PK-A; (3), the effect of substituting Phe for Val in the peptide Arg-Arg-Ala-Ser-Val-Ala, which improves the efficiency of phosphorylation by PK-A (lowering the K_m 4-fold), but has no effect on phosphorylation by ISPK-1. These differences in peptide substrate specificity may account in part for the different rates of phosphorylation of physiological substrates for ISPK-1 and PK-A, such as the G subunit of protein phosphatase-1.     

Comparative specificity of microbial acid proteinases for synthetic peptides. 3. Relationship with their trypsinogen activating ability

The specificities of acid proteinases from Aspergillus niger, Aspergillus saitoi, Rhizopus chinensis, Mucor miehei, Rhodotorula glutinis, and Cladosporium sp., and that of swine pepsin, were determined and compared with ability of the enzymes to activate trypsinogen. Various oligopeptides containing l-lysine, Z-Lys-X-Ala, Z-Lys-(Ala)_m, Z-Lys-Leu-(Ala)₂, and Z-(Ala)_n-Lys-(Ala)₃ (X = various amino acid residues, m = 1–4, n = 1–2) were used as substrates. Of the enzymes which are able to activate trypsinogen, most split these peptides at the peptide bond formed by the carbonyl group of l-lysine. For the peptides to be susceptible to the enzymes it was essential that the chain extended for two or three amino acid residues on the C-terminal side of the catalytic point, and that a bulky or hydrophobic amino acid residue formed the imino-side of the splitting point. The rate of hydrolysis was markedly accelerated by elongation of the peptide chain with l-alanine on the N-terminal side of the catalytic point. Thus, of the substrates used, Z-(Ala)₂-Lys-(Ala)₃ was the most susceptible to the microbial acid proteinases possessing trypsinogen activating ability. On the other hand, M. miehei enzyme and pepsin, which do not activate trypsinogen, showed very little peptidase activity on the peptides.     

Comparative specificity of microbial acid proteinases for synthetic peptides. II. Effect of secondary interaction

To investigate the effect of “secondary interaction” on hydrolysis by various acid proteinases from molds and yeasts, synthetic peptides

amino acid residues) were used as substrates. Pepsin was used for the comparative study. These peptides were split at the peptide bonds indicated by the arrows, permitting examination of the effect of residue X distant by two or three amino acid residues from the hydrolytic site in the peptides. According to the system of Schechter and Berger (Biochem. Biophys. Res. Commun. 27; 157, 1967), the amino acid residues in peptide substrates were numbered P₁, P₂, etc. toward the N-terminal direction from the site of hydrolysis, and P₁′, P₂′, etc. toward the C-terminal direction. The results indicated that hydrolysis by these microbial enzymes is affected by at least six amino acid residues (P₁-P₃ and P₁′-P₃′) in peptide substrates, as is seen with pepsin. Elongation of the peptide chain with suitable amino acid residues from P₁ to P₂ or P₃ and from P₁′ to P₂′ or P₃′ in peptide substrates resulted in much or less increase of hydrolysis depending upon the species of the enzyme producers.     

Comparison of the Enzymatic and Functional Properties of Three Cytosolic Carboxypeptidase Family Members

Nna1 (CCP1) defines a subfamily of M14 metallocarboxypeptidases (CCP1–6) and is mutated in pcd (Purkinje cell degeneration) mice. Nna1, CCP4, and CCP6 are involved in the post-translational process of polyglutamylation, where they catalyze the removal of polyglutamate side chains. However, it is unknown whether these three cytosolic carboxypeptidases share identical enzymatic properties and redundant biological functions. We show that like Nna1, purified recombinant CCP4 and CCP6 deglutamylate tubulin, but unlike Nna1, neither rescues Purkinje cell degeneration in pcd mice, indicating that they do not have identical functions. Using biotin-based synthetic substrates, we established that the three enzymes are distinguishable based upon individual preferences for glutamate chain length, the amino acid immediately adjacent to the glutamate chain, and whether their activity is enhanced by nearby acidic amino acids. Nna1 and CCP4 remove the C-terminal glutamate from substrates with two or more glutamates, whereas CCP6 requires four or more glutamates. CCP4 behaves as a promiscuous glutamase, with little preference for chain length or neighboring amino acid composition. Besides glutamate chain length dependence, Nna1 and CCP6 exhibit higher k_cat/K_m when substrates contain nearby acidic amino acids. All cytosolic carboxypeptidases exhibit a monoglutamase activity when aspartic acid precedes a single glutamate, which, together with their other individual preferences for flanking amino acids, greatly increases the potential substrates for these enzymes and the biological processes in which they act. Additionally, Nna1 metabolized substrates mimicking the C terminus of tubulin in a way suggesting that the tyrosinated form of tubulin will accumulate in pcd mice.     

Comprehensive Mutational Analysis Reveals p6Gag Phosphorylation To Be Dispensable for HIV-1 Morphogenesis and Replication

The structural polyprotein Gag of human immunodeficiency virus type 1 (HIV-1) is necessary and sufficient for formation of virus-like particles. Its C-terminal p6 domain harbors short peptide motifs that facilitate virus release from the plasma membrane and mediate incorporation of the viral Vpr protein. p6 has been shown to be the major viral phosphoprotein in HIV-1-infected cells and virions, but the sites and functional relevance of p6 phosphorylation are not clear. Here, we identified phosphorylation of several serine and threonine residues in p6 in purified virus preparations using mass spectrometry. Mutation of individual candidate phosphoacceptor residues had no detectable effect on virus assembly, release, and infectivity, however, suggesting that phosphorylation of single residues may not be functionally relevant. Therefore, a comprehensive mutational analysis was conducted changing all potentially phosphorylatable amino acids in p6, except for a threonine that is part of an essential peptide motif. To avoid confounding changes in the overlapping pol reading frame, mutagenesis was performed in a provirus with genetically uncoupled gag and pol reading frames. An HIV-1 derivative carrying 12 amino acid changes in its p6 region, abolishing all but one potential phosphoacceptor site, showed no impairment of Gag assembly and virus release and displayed only very subtle deficiencies in viral infectivity in T-cell lines and primary lymphocytes. All mutations were stable over 2 weeks of culture in primary cells. Based on these findings, we conclude that phosphorylation of p6 is dispensable for HIV-1 assembly, release, and infectivity in tissue culture.     

Different specificities of spleen tyrosine protein kinases for synthetic peptide substrates

20 synthetic peptides, each of which includes a tyrosyl residue flanked by either neutral or acidic amino acids in different proportions and at variable positions, have been employed as model substrates for investigation of the site specificity of three tyrosine protein kinases previously isolated from spleen [Brunati, A. M. & Pinna, L. A. (1988) Eur. J. Biochem. 172, 451-457] and conventionally termed TPK-I, TPK-IIB and TPK-III. Comparison of the phosphorylation efficiencies shows that each tyrosine protein kinase is considerably different from the others in both the stringency and the nature of its specificity determinants. By considering, in particular, the kinetic constants obtained with the pentapeptides AAYAA, EEYAA, AEYAA, EAYAA, with the tetrapeptides AYAA and EYAA and with the tripeptides AYA and EYA, it turns out that N-terminal acidic residue(s) are only essential with TPK-IIB for efficient phosphorylation with multiple residues displaying a synergistic effect. The very similar Km (130 microM) but 14-fold-different Vmax values with YEEEEE vs. EEEEEY indicate that an N-terminal rather than C-terminal location of acidic residues is required for a high phosphorylation rate with, though not for binding to TPK-IIB. Acidic residues decrease the phosphorylation rate with TPK-I, a kinase related to the src family which is immunologically indistinguishable from the lyn TPK; they are nearly ineffective, however, with TPK-III, the least specific of the tyrosine protein kinases, which exhibits appreciable activity toward tripeptides and dipeptides like GAY and AY which are not significantly affected by TPK-I and TPK-IIB. While the peptide substrate specificity of TPK-I is similar to that of TPK-IIA, a spleen tyrosine protein kinase previously considered [Brunati, A. M., Marchiori, F., Ruzza, P., Calderan, A., Borin, G. & Pinna, L. A. (1989) FEBS Lett. 254, 145-149], the remarkable requirement of TPK-IIB alone for acidic peptides may suggest the involvement of this enzyme, which is also unique in its failure to autophosphorylate, in the phosphorylation of the highly conserved and quite acidic phosphoacceptor sites of the src family protein kinases.     

Exploring sequence requirements for C₃/C₄ carboxylate recognition in the Pseudomonas aeruginosa cephalosporinase: Insights into plasticity of the AmpC β-lactamase

In Pseudomonas aeruginosa, the chromosomally encoded class C cephalosporinase (AmpC β-lactamase) is often responsible for high-level resistance to β-lactam antibiotics. Despite years of study of these important β-lactamases, knowledge regarding how amino acid sequence dictates function of the AmpC Pseudomonas-derived cephalosporinase (PDC) remains scarce. Insights into structure-function relationships are crucial to the design of both β-lactams and high-affinity inhibitors. In order to understand how PDC recognizes the C₃/C₄ carboxylate of β-lactams, we first examined a molecular model of a P. aeruginosa AmpC β-lactamase, PDC-3, in complex with a boronate inhibitor that possesses a side chain that mimics the thiazolidine/dihydrothiazine ring and the C₃/C₄ carboxylate characteristic of β-lactam substrates. We next tested the hypothesis generated by our model, i.e. that more than one amino acid residue is involved in recognition of the C₃/C₄ β-lactam carboxylate, and engineered alanine variants at three putative carboxylate binding amino acids. Antimicrobial susceptibility testing showed that the PDC-3 β-lactamase maintains a high level of activity despite the substitution of C₃/C₄ β-lactam carboxylate recognition residues. Enzyme kinetics were determined for a panel of nine penicillin and cephalosporin analog boronates synthesized as active site probes of the PDC-3 enzyme and the Arg349Ala variant. Our examination of the PDC-3 active site revealed that more than one residue could serve to interact with the C₃/C₄ carboxylate of the β-lactam. This functional versatility has implications for novel drug design, protein evolution, and resistance profile of this enzyme.     

Kinetic studies of the binding of inhibitors to thermolysin

The K_I values for inhibition of thermolysin activity by N-β-phenylpropionyl-aliphatic amino acids (Gly, Ala, Val, Leu, Ile) are correlated by π, the hydrophobic substituent parameter for the amino acid side chain (log K_I = ?0.73π ?1.80, correlation coefficient = 0.990). By contrast, the K_I values for the corresponding benzyloxycarbonyl amino acids are poorly correlated by π, but show a good correlation with the steric parameter E_s(log K_I = 0.880E_s ? 3.086, correlation coefficient = 0.985). Binding of β-phenylpropionyl-l-alanine is associated with an acidic residue of pK 7.3 and a basic residue of pK 8.0 in the E · I complex, and appears to raise the pK of Glu-143 by 2 units. Binding of benzyloxycarbonyl-Ala and -Phe is associated with an acidic residue of pK 8.0 and two basic residues, both with pK 8.3. Three similar pK values are observed with benzyloxycarbonyl-Phe. These results are interpreted in terms of different modes of binding of β-phenylpropionyl and benzyloxycarbonyl inhibitors.     

Determinants for substrate phosphorylation by Dictyostelium myosin II heavy chain kinases A and B and eukaryotic elongation factor-2 kinase

The alpha kinases are a widespread family of atypical protein kinases characterized by a novel type of catalytic domain. In this paper the peptide substrate recognition motifs for three alpha kinases, Dictyostelium discoideum myosin heavy chain kinase (MHCK) A and MHCK B and mammalian eukaryotic elongation factor-2 kinase (eEF-2K), were characterized by incorporating amino acid substitutions into a previously identified MHCK A peptide substrate (YAYDTRYRR) (Luo X. et al. (2001) J. Biol. Chem. 276, 17836-43). A lysine or arginine in the P+1 position on the C-terminal side of the phosphoacceptor threonine (P site) was found to be critical for peptide substrate recognition by MHCK A, MHCK B and eEF-2K. Phosphorylation by MHCK B was further enhanced 8-fold by a basic residue in the P+2 position whereas phosphorylation by MHCK A was enhanced 2- to 4-fold by basic residues in the P+2, P+3 and P+4 positions. eEF-2K required basic residues in both the P+1 and P+3 positions to recognize peptide substrates. eEF-2K, like MHCK A and MHCK B, exhibited a strong preference for threonine as the phosphoacceptor amino acid. In contrast, the Dictyostelium VwkA and mammalian TRPM7 alpha kinases phosphorylated both threonine and serine residues. The results, together with a phylogenetic analysis of the alpha kinase catalytic domain, support the view that the metazoan eEF-2Ks and the Dictyostelium MHCKs form a distinct subgroup of alpha kinases with conserved properties.     

Multimodal Mechanism of Action for the Cdc34 Acidic Loop: A CASE STUDY FOR WHY UBIQUITIN-CONJUGATING ENZYMES HAVE LOOPS AND TAILS*

Together with ubiquitin ligases (E3), ubiquitin-conjugating enzymes (E2) are charged with the essential task of synthesizing ubiquitin chains onto protein substrates. Some 75% of the known E2s in the human proteome contain unique insertions in their primary sequences, yet it is largely unclear what effect these insertions impart on the ubiquitination reaction. Cdc34 is an important E2 with prominent roles in cell cycle regulation and signal transduction. The amino acid sequence of Cdc34 contains an insertion distal to the active site that is absent in most other E2s, yet this acidic loop (named for its four invariably conserved acidic residues) is critical for Cdc34 function both in vitro and in vivo. Here we have investigated how the acidic loop in human Cdc34 promotes ubiquitination, identifying two key molecular events during which the acidic loop exerts its influence. First, the acidic loop promotes the interaction between Cdc34 and its ubiquitin ligase partner, SCF. Second, two glutamic acid residues located on the distal side of the loop collaborate with an invariably conserved histidine on the proximal side of the loop to suppress the pK_a of an ionizing species on ubiquitin or Cdc34 which greatly contributes to Cdc34 catalysis. These results demonstrate that insertions can guide E2s to their physiologically relevant ubiquitin ligases as well as provide essential modalities that promote catalysis.     

Involvement of Val 315 located in the C-terminal region of thermolysin in its expression in Escherichia coli and its thermal stability

Thermolysin is a thermophilic and halophilic zinc metalloproteinase that consists of β-rich N-terminal (residues 1–157) and α-rich C-terminal (residues 158–316) domains. Expression of thermolysin variants truncated from the C-terminus was examined in E. coli culture. The C-terminal Lys316 residue was not significant in the expression, but Val315 was critical. Variants in which Val315 was substituted with fourteen amino acids were prepared. The variants substituted with hydrophobic amino acids such as Leu and Ile were almost the same as wild-type thermolysin (WT) in the expression amount, α-helix content, and stability. Variants with charged (Asp, Glu, Lys, and Arg), bulky (Trp), or small (Gly) amino acids were lower in these characteristics than WT. All variants exhibited considerably high activities (50–100% of WT) in hydrolyzing protein and peptide substrates. The expression amount, helix content, and stability of variants showed good correlation with hydropathy indexes of the amino acids substituted for Val315. Crystallographic study of thermolysin has indicated that V315 is a member of the C-terminal hydrophobic cluster. The results obtained in the present study indicate that stabilization of the cluster increases thermolysin stability and that the variants with higher stability are expressed more in the culture. Although thermolysin activity was not severely affected by the variation at position 315, the stability and specificity were modified significantly, suggesting the long-range interaction between the C-terminal region and active site.     

Substrate specificity determinants for casein kinase II as deduced from studies with synthetic peptides

The specificity of casein kinase II has been further defined by analyzing the kinetics of phosphorylation reactions using a number of different synthetic peptides as substrates. The best peptide substrates are those in which multiple acidic amino acids are present on both sides of the phosphorylatable serine or threonine. Acidic residues on the NH2-terminal side of the serine (threonine) greatly enhance the kinetic constants but are not absolutely required. Acidic residues on the COOH-terminal side of the serine (threonine) are absolutely required. One position for which the occupation of an acidic residue is especially critical is the position located 3 residues to the COOH terminus of the phosphate acceptor site, although the presence of an acidic amino acid in the positions that are 4 or 5 residues removed may also provide an appropriate structure that will serve as a substrate for the kinase. Aspartate serves as a better amino acid determinant than glutamate. A relatively short sequence of amino acids surrounding the phosphate acceptor site appears to serve as the basis for the specificity of casein kinase II. The peptides in this study were also assayed with casein kinase I and the casein kinase from the mammary gland so that the specificities of these kinases could be compared to that of casein kinase II.     

Comparative specificity of microbial acid proteinases for synthetic peptides. Primary specificity with Z-tetrapeptides

The substrates Z-X

Leu-(Ala)₂ and

Z-Phe X-(Ala)₂ (Z = benzyloxycarbonyl, X = various amino acid residues) were synthesized in order to investigate the primary specificity of acid proteinases from molds and yeasts. Since these peptides are mainly susceptible to cleavage by the enzymes at the peptide bonds shown by the arrows, it was possible to determine the specificity with respect to the amino acid residues on both sides of the splitting point. Pepsin was used for comparison. The results indicated that the microbial acid proteinases exhibit specificity for aromatic or hydrophobic amino acid residues on both sides of splitting point in peptide substrates, as does pepsin. However, the microbial enzymes showed somewhat broader specificity than pepsin. The former enzymes, which possess trypsinogen-activating ability, show specificity for a lysine residue, while pepsin or Mucor rennin-like enzyme does not. Although pepsin is very specific for a tyrosine residue on the imino side of the splitting point, the microbial enzymes do not show such stringency.     

Effect of secondary interaction on the enzymatic activity of trypsin-like enzymes from Streptomyces

The effect of secondary interaction with substrate on the enzymatic activity of trypsin-like enzymes from Streptomyces was studied using Z-Lys-(Ala)_m, Z-(Ala)_nLys-OMe, Z-Lys-X-Ala and Z-X-Lys-OMe (m = 1–4; n = 0–2; X = various amino acid residues) as substrates and a comparison was made with bovine trypsin. These peptides are susceptible to cleavage at the peptide or ester bonds containing the carbonyl group of l-lysine, which enabled determination of the effect of chain-length on either side of the sensitive l-lysine residue in the first two types of peptide, and the effect of side-chains of the amino acid residues immediately neighboring on either side of the sensitive l-lysine residue in the latter two types of peptide. The results indicate that the enzymatic activity of the trypsin-like enzymes are little affected by secondary interaction, similarly as seen with bovine trypsin.     

Aeromonas neutral protease: Specificity toward extended substrates

Kinetic measurements on the action of Aeromonas neutral protease toward blocked peptide substrates were made in order to determine the most favorable fit on the enzyme subsites that bind the residues flanking the scissile bond and to define the number of secondary sites involved in catalysis. Variations in the identity of P₁′,³ the residue furnishing the amino group to the scissile bond, produced significant changes in k_rmcat, whereas the identity of P₁′, the residue donating the carboxyl group, was of much less catalytic importance. Comparison of these results with those of previous investigators of other bacterial neutral proteases indicated distinct differences in specificity of the Aeromonas enzyme and revealed that phenylalanyl residues, rather than leucyl, were preferred in the P₁′ position. Additional binding sites on the carboxyl side of the scissile bond were shown to be important to catalytic efficiency and it is evident that at least three residues (P₁t$\text{,}\text{?}$ P₂′, P₃′) are involved while only two residues (P₂, P₁) on the amino terminal side of the sensitive bond are implicated.     

Comparative study of various serine alkaline proteinases from microorganisms. Esterase activity against N-acylated peptide ester substrates

Hydrolyses of N-acylated peptide ester substrates by various serine alkaline proteinases from bacterial and mold origin were compared using Ac- or Z-(Ala)_m-X-OMe (m = 0-2 or 0-3; X = phenylalanine, alanine, and lysine) as esterase substrates. The results indicated that the esterase activities of these enzymes were markedly promoted by elongating the peptide chain from P₁ to P₂ or P₃ with alanine, irrespective of the kind of the amino acid residue at the P₁-position (amino acid residues in peptide substrates are numbered according to the system of Schechter and Berger (1)). The effect of the kind of amino acid residue at the P₂-position was further determined using Z-X-Lys-OMe (X = glycine, alanine, leucine, or phenylalanine) as esterase substrates. Alanine was the most efficient residue as X with subtilisins and Streptomyces fradiae Ib enzyme, while leucine or phenylalanine were most efficient with the enzymes from Streptomyces fradiae II, Aspergillus sojae, and Aspergillus melleus. All the serine alkaline proteinases tested in this study were sensitive to Z-Ala-Gly-PheCH₂Cl, the dependence of inhibition on the inhibitor concentration differed among the enzymes.     

Deciphering the Arginine-binding preferences at the substrate-binding groove of Ser/Thr kinases by computational surface mapping

Protein kinases are key signaling enzymes that catalyze the transfer of γ-phosphate from an ATP molecule to a phospho-accepting residue in the substrate. Unraveling the molecular features that govern the preference of kinases for particular residues flanking the phosphoacceptor is important for understanding kinase specificities toward their substrates and for designing substrate-like peptidic inhibitors. We applied ANCHORSmap, a new fragment-based computational approach for mapping amino acid side chains on protein surfaces, to predict and characterize the preference of kinases toward Arginine binding. We focus on positions P-2 and P-5, commonly occupied by Arginine (Arg) in substrates of basophilic Ser/Thr kinases. The method accurately identified all the P-2/P-5 Arg binding sites previously determined by X-ray crystallography and produced Arg preferences that corresponded to those experimentally found by peptide arrays. The predicted Arg-binding positions and their associated pockets were analyzed in terms of shape, physicochemical properties, amino acid composition, and in-silico mutagenesis, providing structural rationalization for previously unexplained trends in kinase preferences toward Arg moieties. This methodology sheds light on several kinases that were described in the literature as having non-trivial preferences for Arg, and provides some surprising departures from the prevailing views regarding residues that determine kinase specificity toward Arg. In particular, we found that the preference for a P-5 Arg is not necessarily governed by the 170/230 acidic pair, as was previously assumed, but by several different pairs of acidic residues, selected from positions 133, 169, and 230 (PKA numbering). The acidic residue at position 230 serves as a pivotal element in recognizing Arg from both the P-2 and P-5 positions.     

Specificity,anchoring, and subsites in the active center of a microvillar aminopeptidase purified from Tenebrio molitor (Coleoptera) midgut cells

There is a single membrane-bound aminopeptidase (AP) in Tenebrio molitor L. larval midguts with a pH optimum of 8.0. This enzyme is restricted to the posterior third of the midgut, where it accounts for about 55% of the microvillar proteins. AP, after being solubilized in detergent or released by papain, was purified to homogeneity. The enzyme is a glycoprotein rich in mannose residues. N-terminal sequencing of papain and detergent forms of AP resulted in the same sequence containing the common motif YRLP. These and other data, which included partition in Triton X-114 and incubation with glycosyl-phosphatidylinositol (GPI)-specific phospholipase C and GPI-specific phospholipase D suggest that AP (Mr 90 000) is inserted into the microvillar membranes by a C-terminal anchor, which is a peptide or a papain — released protected GPI anchor. AP has a broad specificity towards the N-terminal amino acid residue of substrates, although it does not hydrolyze acidic aminoacyl-peptides, thus resembling mammalian aminopeptidase N (EC 3.4.11.2). k_cat/K_m ratios obtained for different di-, tri-, tetra-, and pentapeptides suggest that there are four subsites in AP, and that subsites S₁, S₁′ and S₂′ are pockets able to bind bulky aminoacyl residues. This hypothesis agrees with the fact that amastatin is a stronger inhibitor of AP than bestatin. Amastatin is a slow, tight-binding inhibitor of AP. Bestatin binds in a rapidly reversible mode in S₁′ and S₂′ subsites of AP.