Dicyclohexylcarbodiimide cross-links two conserved residues, Asp-184 and Lys-72, at the active site of the catalytic subunit of cAMP-dependent protein kinase

In the absence of MgATP, the catalytic subunit of cAMP-dependent protein kinase is irreversibly inhibited by the hydrophobic carbodiimide dicyclohexylcarbodiimide, and this inhibition is most likely due to the formation of a cross-link between a carboxyl group and a lysine residue in the active site (Toner-Webb & Taylor, 1987). In order to identify these cross-linked residues, the catalytic subunit was modified by dicyclohexylcarbodiimide and then treated with acetic anhydride and digested with trypsin. The resulting peptides were resolved by high-performance liquid chromatography. One major absorbing tryptic peptide and one smaller peptide consistently and reproducibly showed a decrease in absorbance after the catalytic subunit had been treated with DCCD. These peptides correspond to residues 166-190 and 57-93, respectively. A unique peptide was isolated from the modified catalytic subunit, and the sequence of this peptide established that the cross-linking occurred between Asp-184 and Lys-72. The cross-linking of these two residues, which were both identified previously as essential residues, confirms the likelihood that each plays a role in the functioning of this enzyme. The fact that Asp-184 and Lys-72 appear to be invariant in all protein kinases further supports the hypothesis that these two residues, located close to one another at the active site of the enzyme, play essential roles in catalysis.     

Recombinant rat guanidinoacetate methyltransferase: Study of the structure by trace labeling lysine residues with acetic anhydride

• 1.1. The reactivities of lysine residues of recombinant rat guanidinoacetate methyltransferase were determined by trace labeling with acetic anhydride.
• 2.2. Lys-113 and -160 were weakly reactive and Lys-178 and -234 were unreactive toward the reagent. The six lysines (Lys-38, -83, -104, -108, -152 and -180) showed moderate reactivities. The N-terminal amino group was very reactive.
• 3.3. S-Adenosylmethionine did not alter the reactivities of lysines significantly, but the reactivity of Lys-38 was substantially reduced in the presence of S-adenosylmethionine and guanidinoacetate.

     

Differential labeling of the catalytic subunit of cAMP-dependent protein kinase with a water-soluble carbodiimide: identification of carboxyl groups protected by MgATP and inhibitor peptides

The catalytic subunit of cAMP-dependent protein kinase typically phosphorylates protein substrates containing basic amino acids preceding the phosphorylation site. To identify amino acids in the catalytic subunit that might interact with these basic residues in the protein substrate, the enzyme was treated with a water-soluble carbodiimide, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), in the presence of [14C]glycine ethyl ester. Modification of the catalytic subunit in the absence of substrates led to the irreversible, first-order inhibition of activity. Neither MgATP nor a 6-residue inhibitor peptide alone was sufficient to protect the catalytic subunit against inactivation by the carbodiimide. However, the inhibitor peptide and MgATP together completely blocked the inhibitory effects of EDC. Several carboxyl groups in the free catalytic subunit were radiolabeled after the catalytic subunit was modified with EDC and [14C]glycine ethyl ester. After purification and sequencing, these carboxyl groups were identified as Glu 107, Glu 170, Asp 241, Asp 328, Asp 329, Glu 331, Glu 332, and Glu 333. Three of these amino acids, Glu 331, Glu 107, and Asp 241, were labeled regardless of the presence of substrates, while Glu 333 and Asp 329 were modified to a slight extent only in the free catalytic subunit. Glu 170, Asp 328, and Glu 332 were all very reactive in the apoenzyme but fully protected from modification by EDC in the presence of MgATP and an inhibitor peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential reactivities of lysines in calmodulin complexed to phosphatase

Calmodulin and calmodulin complexed with calcineurin phosphatase were trace labeled with [3H]acetic anhydride and the incorporation of [3H]acetate into each epsilon-amino lysine of calmodulin was measured. The relative reactivities of calmodulin lysines were higher in the presence of Ca2+ than in the presence of EGTA, and the order was: Lys-75 greater than Lys-94 greater than Lys-148 greater than or equal to Lys-77 greater than Lys-13 greater than or equal to Lys-21 greater than Lys-30. The changes in relative reactivity implied a change in conformation. When calmodulin was complexed with the phosphatase, Lys-21, Lys-77, and Lys-148 were most protected, implying that these residues are at or near the interaction sites or are conformationally perturbed by the interaction. Lys-30 and Lys-75 were slightly protected, lysine 13 showed no change, while lysine 94 significantly increased in reactivity. Comparison with results obtained from myosin light chain kinase using a similar technique (Jackson, A. E., Carraway, K. L., III, Puett, D., and Brew, K. (1986) J. Biol. Chem. 261, 12226-12232) reveals that calmodulin may interact with each of the two enzymes similarly at or near Lys-21, Lys-75, and Lys-148; one difference with phosphatase is that complex formation also involved Lys-77. These findings suggest that calmodulin interacts differently with its target enzymes.     

Sites of protein-protein interaction on the mitochondrial F1-ATPase inhibitor protein.

We have investigated the structure of the mitochondrial F1-ATPase inhibitor protein from ox heart by using a differential trace-labelling method. This method has also been used to determine sites on the inhibitor protein involved in binding to both the isolated mitochondrial ATPase (F1) and to a specific anti-inhibitor antibody. Native, free inhibitor was trace-labelled on its lysine and serine residues with [14C]acetic anhydride, and inhibitor protein unfolded in guanidinium chloride or specifically bound to another protein, with [3H]acetic anhydride. Exposure/concealment of residues was deduced from the 14C/3H ratios of the peptides in a proteolytic digest of the inhibitor, after separation by h.p.l.c. None of the lysine or serine residues in the native inhibitor are as exposed as in the unfolded form. There is a gradient of reactivity, with residues 54-58 being most concealed and exposure increasing towards either end of the protein. A slight decrease in reactivity is noted in residues 1-3, suggesting that the N-terminus may be in a fairly restricted environment. These findings are discussed in the light of the predicted structure of the inhibitor protein. All but one of the labelled residues increases in reactivity when inhibitor protein binds to F1. The exception, Lys-24, is only slightly concealed. Hence, F1 binding appears not to involve the lysine or serine residues directly. This finding is consistent with the view that the F1-inhibitor interaction is hydrophobic in nature. Complementary information was provided using an anti-inhibitor antibody that binds to a site on the inhibitor different from that at which F1 binds. Binding of this antibody conceals residues 54, 58, and 65 considerably. This confirms that F1 does not interact with these hydrophilic residues on the inhibitor protein.     

Lysine reactivities of tropomyosin complexed with troponin

The relative reactivities of lysine residues of tropomyosin complexed with troponin have been measured in order to locate the binding site of troponin on tropomyosin in a complex between the two native proteins. The lysines were labeled with acetic anhydride using a competitive labeling procedure and the relative reactivities of tropomyosin lysine containing peptides were compared to those from tropomyosin labeled in the absence of troponin (S. E. Hitchcock-DeGregori, S. F. Lewis, and T. M.-T. Chou, (1985) Biochemistry 24, 3305-3314). Analysis of about two-thirds of the lysines indicates that troponin affects the reactivities of lysines along the length of the tropomyosin, indicating long-range effects. The inferred binding site is more extensive than previously reported, about 25 nm, extending from res. 136 to the carboxy-terminus and to res. 30 beyond the end-to-end overlap in the amino-terminal region of the next tropomyosin molecule.     

Novel mechanism of surface catalysis of protein adduct formation. NMR studies of the acetylation of ubiquitin

Reactivity of surface lysyl residues of proteins with a broad range of chemical agents has been proposed to be dependent on the catalytic microenvironment of the residue. We have investigated the acetylation of wild type ubiquitin and of the UbH68N mutant to evaluate the potential contribution of His-68 to the reactivity of Lys-6, which is about 4 A distant. These studies were performed using [1-(13)C]acetyl salicylate or [1,1'-(13)C(2)]acetic anhydride, and the acetylated products were detected by two-dimensional heteronuclear multiple quantum coherence spectroscopy. The results demonstrate that His-68 makes a positive contribution to the rate of acetylation of Lys-6 by labeled aspirin. Additionally, a pair of transient resonances is observed after treatment of wild type ubiquitin with the labeled acetic anhydride but not upon treatment of the H68N mutant. These resonances are assigned to the acetylated His-68 residue. The loss of intensity of the acetylhistidine resonances is accompanied by an increase in intensity of the acetyl-Lys-6 peak, supporting the existence of a transacetylation process between the acetylhistidine 68 and lysine 6 residues located on the protein surface. Hence, this may be the first direct demonstration of a catalytic intermediate forming on the protein surface.     

Tropomyosin lysine reactivities and relationship to coiled-coil structure

We have carried out a detailed analysis of tropomyosin structure using lysines as specific probes for the protein surface in regions of the molecule that have not been investigated by other methods. We have measured the relative reactivities of lysines in rabbit skeletal muscle alpha, alpha-tropomyosin with acetic anhydride using a competitive labeling procedure. We have identified 37 of 39 lysines and find that they range 20-fold in reactivity. The observed reactivities are related to the coiled-coil model of the tropomyosin molecule [Crick, F.H.C. (1953) Acta Crystallogr. 6, 689-697; McLachlan, A.D., Stewart, M., & Smillie, L.B. (1975) J. Mol. Biol. 98, 281-291] and other available chemical and physical information about the structure. In most cases, the observed lysine reactivities can be explained by allowable interactions with neighboring amino acid side chains on the same or facing alpha-helix. However, we found no correlation between reactivity and helical position of a given lysine. For example, lysines in the outer helical positions included lysines of low as well as high reactivity, indicating that they vary widely in their accessibility to solvent and that the coiled coil is heterogeneous along its length. Furthermore, the middle of the molecule (residues 126-182) that is susceptible to proteolysis and known to be the least stable region of the protein also contains some of the least and most reactive lysines. We have discussed the implications of our results on our understanding the structures of tropomyosin and other coiled-coil proteins as well as globular proteins containing helical regions.     

Identification of aspartate-184 as an essential residue in the catalytic subunit of cAMP-dependent protein kinase

The hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD) was previously shown to be an irreversible inhibitor of the catalytic subunit of cAMP-dependent protein kinase, and MgATP protected against inactivation [Toner-Webb, J., & Taylor, S. S. (1987) Biochemistry 26, 7371]. This inhibition by DCCD indicated that an essential carboxyl group was present at the active site of the enzyme even though identification of that carboxyl group was not possible. This presumably was because a nucleophile on the protein cross-linked to the electrophilic intermediate formed when the carbodiimide reacted with the carboxyl group. To circumvent this problem, the catalytic subunit first was treated with acetic anhydride to block accessible lysine residues, thus preventing intramolecular cross-linking. The DCCD reaction then was carried out in the presence of [14C]glycine ethyl ester in order to trap any electrophilic intermediates that were generated by DCCD. The modified protein was treated with trypsin, and the resulting peptides were separated by HPLC. Two major radioactive peptides were isolated as well as one minor peptide. MgATP protected all three peptides from covalent modification. The two major peaks contained the same modified carboxyl group, which corresponded to Asp-184. The minor peak contained a modified glutamic acid, Glu-91. Both of these acidic residues are conserved in all protein kinases, which is consistent with their playing essential roles. The positions of Asp-184 and Glu-91 have been correlated with the overall domain structure of the molecule. Asp-184 may participate as a general base catalyst at the active site. A third carboxyl group, Glu-230, also was identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

The reactive site of eggplant trypsin inhibitor.

The reactive site peptide bond of the eggplant inhibitor against trypsin [EC 3.4.21.4] was identified by chemical modifications with 1,2-cyclohexanedione, 2,4,6-trinitrobenzenesulfonic acid, acetic anhydride and glyoxal, and by sequential treatments with trypsin and carboxypeptidase B [EC 3.4.12.3]. The inhibitor was significantly inactivated by chemical modifications of arginine residues, but was not affected by lysine modifications. Free arginine was released from the trypsin-modified inhibitor by carboxypeptidase B digestion, accompanied by a marked loss of inhibitory activity. A serine residue was newly exposed at the N-terminal amino acid of the inhibitor after modification with trypsin. The reactive site of the inhibitor against trypsin was concluded to be an arginylseryl bond. The inhibitor was completely inactivated by full reduction of its disulfide bonds.     

MgATP-induced conformational change of the catalytic subunit of cAMP-dependent protein kinase

Conformational changes of the cAMP-dependent protein kinase (PKA) catalytic (C) subunit are critical for the catalysis of gamma-phosphate transfer from adenosine 5'-triphosphate (ATP) to target proteins. Time-resolved fluorescence anisotropy (TRFA) was used to investigate the respective roles of Mg(2+), ATP, MgATP, and the inhibitor peptide (IP20) in the conformational changes of a 5,6-carboxyfluorescein succinimidyl ester (CF) labeled C subunit ((CF)C). TRFA decays were fit to a biexponential equation incorporating the fast and slow rotational correlation times phi(F) and phi(S). The (CF)C apoenzyme exhibited the rotational correlation times phi(F)=1.8+/-0.3 ns and phi(S)=20.1+/-0.6 ns which were reduced to phi(F)=1.1+/-0.2 ns and phi(S)=13.3+/-0.9 ns in the presence of MgATP. The reduction in rotational correlation times indicated that the (CF)C subunit adopted a more compact shape upon formation of a (CF)C.MgATP binary complex. Neither Mg(2+) (1-3 mM) nor ATP (0.4 mM) alone induced changes in the (CF)C subunit conformation equivalent to those induced by MgATP. The effect of MgATP was removed in the presence of ethylenediaminetetraacetic acid (EDTA). The addition of IP20 and MgATP to form the (CF)C x MgATP x IP20 ternary complex produced rotational correlation times similar to those of the (CF)C x MgATP binary complex. However, IP20 alone did not elicit an equivalent reduction in rotational correlation times. The results indicate that binding of MgATP to the C subunit may induce conformation changes in the C subunit necessary for the proper stereochemical alignment of substrates in the subsequent phosphorylation.     

Study of the structure of troponin-C by measuring the relative reactivities of lysines with acetic anhydride

The structure of troponin-C² has been studied by measuring the relative reactivity of lysines with acetic anhydride using a competitive labeling method. Troponin-C was acetylated free and complexed with troponin-I and -T in the native state with [³H]acetic anhydride and combined with [¹⁴C]troponin-C that had been acetylated in 6 m-guanidine · HCl. Peptides containing labeled lysines were isolated following chymotryptic and tryptic digestion and identified in the published sequence. The ${}^3\text{H}{}^{14}\text{C}$ ratio of these peptides was used as a measure of relative accessibility of the lysines. Troponin-C contains 9 lysine residues. In free troponin-C Lys20 was the least reactive and Lys153 was the most reactive; the remaining 7 had intermediate reactivities. Lys52 was more reactive in the presence of 10^?5m-Ca²⁺ than in 0.2 mm-EGTA (+2 mm-MgCl₂). When troponin-C was labeled in the native troponin complex, Lys20 and 153 were the least and most reactive, respectively. Peptides containing Lys52, (84, 88, 90) and (136, 140) were reduced in reactivity relative to Lys37 and 153, suggesting that these regions are involved in binding to the other troponin components. The reactivities of Lys37 and (136, 140) were influenced by the calcium ion concentration. A similar pattern of reactivities was seen when troponin-C was complexed with troponin-I and complex formation with troponin-T resulted in reduced reactivity of Lys52 and (84, 88, 90). The results are related to structural studies of troponin-C and to the predicted three-dimensional structure based on carp parvalbumin.     

Escherichia coli signal peptide peptidase A is a serine-lysine protease with a lysine recruited to the nonconserved amino-terminal domain in the S49 protease family

Escherichia coli signal peptide peptidase A (SppA) is a serine protease which cleaves signal peptides after they have been proteolytically removed from exported proteins by signal peptidase processing. We present here results of site-directed mutagenesis studies of all the conserved serines of SppA in the carboxyl-terminal domain showing that only Ser 409 is essential for enzymatic activity. Also, we show that the serine hydrolase inhibitor FP-biotin inhibits SppA and modifies the protein but does not label the S409A mutant with an alanine substituted for the essential serine. These results are consistent with Ser 409 being directly involved in the proteolytic mechanism. Remarkably, additional site-directed mutagenesis studies showed that none of the lysines or histidine residues in the carboxyl-terminal protease domain (residues 326-549) is critical for activity, suggesting this domain lacks the general base residue required for proteolysis. In contrast, we found that E. coli SppA has a conserved lysine (K209) in the N-terminal domain (residues 56-316) that is essential for activity and important for activation of S409 for reactivity toward the FP-biotin inhibitor and is conserved in those other bacterial SppA proteins that have an N-terminal domain. We also performed alkaline phosphatase fusion experiments that establish that SppA has only one transmembrane segment (residues 29-45) with the C-terminal domain (residues 46-618) protruding into the periplasmic space. These results support the idea that E. coli SppA is a Ser-Lys dyad protease, with the Lys recruited to the amino-terminal domain that is itself not present in most known SppA sequences.     

Localization of lysine residues in the binding domain of the K99 fibrillar subunit of enterotoxigenic Escherichia coli

Modification of lysine residues with 4-chloro-3,5-dinitrobenzoate results in the loss of the binding capacity of K99 fibrillae to horse erythrocytes (Jacobs, A.A.C., van Mechelen, J.R. and de Graaf, F.K. (1985) Biochim. Biophys. Acta 832, 148-155). In the present study we used dinitrobenzoate as a spectral probe to map the modified residues. After the incorporation of 0.7 mol CDNB per mol subunit, 90% of the binding activity disappeared and the lysine residues at positions 87, 132 and 133 incorporated 20%, 27.5% and 52.2% of the totally incorporated label, respectively. In the presence of the glycolipid receptor, Lys-132 and Lys-133 were partially protected against modification, while Lys-87 was not protected. The results suggest that Lys-132 and Lys-133 are part of the receptor-binding domain of the K99 fibrillar subunit and that the positive charges on these residues are important for the interaction of the fibrillae with the negatively charged sialic acid residue of the glycolipid receptor. A striking homology was found between a six-amino-acid residue segment of K99, containing Lys-132 and Lys-133, and segments of three other sialic-acid-specific lectins; cholera toxin B subunit, heat-labile toxin B subunit of Escherichia coli and CFA1 fimbrial subunit, suggesting that these segments might also be part of the receptor-binding domain in these three proteins.     

Site-specific glycation of lens crystallins by ascorbic acid.

The oxidation of ascorbic acid leads to the formation of several compounds which are capable of reacting with protein amino groups via a Maillard reaction. Radioactivity from [1-14C]ascorbic acid was linearly incorporated into lens crystallins over a 10 day period in the presence of NaCNBH3. This rate of incorporation was 6-7-fold more rapid than that obtained with [14C]glucose under the same conditions. SDS-PAGE showed a linear incorporation into all the crystallin subunits. [1-14C]Ascorbic acid-label led alpha-crystallin was separated into its component A and B subunits, and each was digested with chymotrypsin. HPLC peptide analysis showed a differential labelling of the various lysine residues. Analysis of the peptides by mass spectrometry allowed the identification of the sites and the extent of modification. These values ranged from 6% for Lys-78 to 36% for Lys-11 in the A subunit and from 5% for Lys-82 to an average of 38% for the peptide containing Lys-166, Lys-174 and Lys-175 in the B subunit. Amino acid analysis demonstrated a single modification reaction producing N epsilon-(carboxymethyl)lysine. This agreed with the mass increase of 58 observed for each modified peptide.     

Contribution of conserved lysine residues in the alpha2-antiplasmin C terminus to plasmin binding and inhibition

α(2)-Antiplasmin is the physiological inhibitor of plasmin and is unique in the serpin family due to N- and C-terminal extensions beyond its core domain. The C-terminal extension comprises 55 amino acids from Asn-410 to Lys-464, and the lysine residues (Lys-418, Lys-427, Lys-434, Lys-441, Lys-448, and Lys-464) within this region are important in mediating the initial interaction with kringle domains of plasmin. To understand the role of lysine residues within the C terminus of α(2)-antiplasmin, we systematically and sequentially mutated the C-terminal lysines, studied the effects on the rate of plasmin inhibition, and measured the binding affinity for plasmin via surface plasmon resonance. We determined that the C-terminal lysine (Lys-464) is individually most important in initiating binding to plasmin. Using two independent methods, we also showed that the conserved internal lysine residues play a major role mediating binding of the C terminus of α(2)-antiplasmin to kringle domains of plasmin and in accelerating the rate of interaction between α(2)-antiplasmin and plasmin. When the C terminus of α(2)-antiplasmin was removed, the binding affinity for active site-blocked plasmin remained high, suggesting additional exosite interactions between the serpin core and plasmin.     

Crucial role of conserved lysine 277 in the fidelity of tRNA aminoacylation by Escherichia coli valyl-tRNA synthetase

Valyl-tRNA synthetase (ValRS) from Escherichia coli undergoes covalent valylation by a donor valyl adenylate synthesized by the enzyme itself. ValRS could also be modified, although to a lesser extent, by the noncognate isosteric substrate L-threonine from a donor threonyl adenylate synthesized by the synthetase itself, or by the nonsubstrate methionine from methionyl adenylate produced by catalytic amounts of methionyl-tRNA synthetase. MALDI mass spectrometry analysis designated lysines 154, 162, 170, 533, 554, 593, 894, 930, and 940 of ValRS as the target residues for the attachment of valine. Following autothreonylation, lysines 162, 170, 178, 277, 291, 554, 580, 593, 861, 894, and 930 were found to be modified. Finally, L-Met-labeled residues were lysines 118, 162, 170, 178, 277, and 938. Alignment of the available ValRS amino acid sequences showed that lysines 277 and 554 are strictly conserved (with the exception concerning replacement of Lys-277 with a methionine or a tyrosine in archaebacteria), suggesting that these residues might be functionally significant. Indeed, lysine 554 of ValRS is the first lysine of the Lys-Met-Ser-Lys-Ser signature of the catalytic site of class I aminoacyl-tRNA synthetases. Lys-277 which is labeled by L-threonine or L-methionine, and not by L-valine, is located at or near the editing site, in the three-dimensional structure of ValRS. The role of lysine 277 was evaluated by site-directed mutagenesis. The Lys277Ala mutant (K277A) exhibited a posttransfer Thr-tRNA(Val) editing rate that was significantly lower than that observed for the wild-type enzyme. In addition, the K277A substitution altered amino acid discrimination in the editing site, resulting in hydrolysis of the correctly charged cognate Val-tRNA(Val). Finally, significant amounts of mischarged Thr-tRNA(Val) were produced by the K277A mutant, and not by wild-type ValRS. Altogether, our results designate Lys-277 as a likely candidate for nucleophilic attack of misacylated tRNA in the editing site of ValRS.     

Reactivity and ionization constants of the lysine residues in apovitellenin i of emu egg yolk low-density lipoprotein by competitive labelling.

Competitive labelling with[14C]acetic anhydride over a range of pH values has been used to explore the surface topography of the apovitellenin I moiety in emu egg yolk low-density lipoprotein. The reaction of the lysine xi-amino groups with acetic anhydride has been related to pH in a set of titration curves; from these, the reactivities relative to alanine and the ionization constants of all but the amino terminal lysines have been determined. All lysines have near normal pKa values around 10, and lower than normal reactivities (except the amino terminal lysine). At pH values above 10, the titration curves show breaks where the epsilon-amino groups become much more reactive, except for lysine 71 which in this regard behaves like a normally ionizing lysine in not showing a discontinuity. Most of the basic residues in this apoprotein may occur clustered at the surface of the molecule. This accounts best for the observed low reactivities and pKa values. The amino terminal lysine residue is presumably completely exposed to the aqueous environment.     

Cross-linking of plasminogen activator inhibitor 2 and alpha 2-antiplasmin to fibrin(ogen)

In this study, we identified lysine residues in the fibrinogen Aalpha chain that serve as substrates during transglutaminase (TG)-mediated cross-linking of plasminogen activator inhibitor 2 (PAI-2). Comparisons were made with alpha(2)-antiplasmin (alpha(2)-AP), which is known to cross-link to lysine 303 of the Aalpha chain. A 30-residue peptide containing Lys-303 specifically competed with fibrinogen for cross-linking to alpha(2)-AP but not for cross-linking to PAI-2. Further evidence that PAI-2 did not cross-link via Lys-303 was the cross-linking of PAI-2 to I-9 and des-alphaC fibrinogens, which lack 100 and 390 amino acids from the C terminus of the Aalpha chain, respectively. PAI-2 or alpha(2)-AP was cross-linked to fibrinogen and digested with trypsin or endopeptidase Glu-C, and the resulting peptides analyzed by mass spectrometry. Peptides detected were consistent with tissue TG (tTG)-mediated cross-linking of PAI-2 to lysines 148, 176, 183, 457 and factor XIIIa-mediated cross-linking of PAI-2 to lysines 148, 230, and 413 in the Aalpha chain. alpha(2)-AP was cross-linked only to lysine 303. Cross-linking of PAI-2 to fibrinogen did not compete with alpha(2)-AP, and the two proteins utilized different lysines in the Aalpha chain. Therefore, PAI-2 and alpha(2)-AP can cross-link simultaneously to the alpha polymers of a fibrin clot and promote resistance to lysis.     

Succinylation of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus. A reactive threonine residue in the apoenzyme.

1. Glyceraldehyde-3-phosphate dehydrogenase from bacillus stearothermophilus can be extensively succinylated in the presence of substrates and coenzyme without appreciable loss of activity. 2. The apoenzyme in the absence of substrates is rapidly inhibited by small amounts of succinic anhydride. NAD+, glyceraldehyde-3-phosphate and inorganic phosphate all afford protection from inhibition, and inhibition is slowly reversed in the presence of pyrophosphate at pH 8.5. 3. Kinetic and spectral studies have shown that the specific inhibition is associated with the succinylation of the aliphatic hydroxyl group of a serine or threonine residue. 4. The residue specifically succinylated has been identified as one of the two threonine residues, most probably Thr-150, adjacent to the activ-site cysteine residue in the primary structure. Its unusual reactivity is discussed in relation to the three-dimensional structure of the enzyme. 5. A second residue, a lysine homologous with Lys-212 in the pig muscle enzyme, can be succinylated in both holoenzyme and apoenzyme with no detectable effect upon the enzymic activity.