Crystal structures of a unique thermal-stable thymidylate synthase from Bacillus subtilis.

Unlike all other organisms studied to date, Bacillus subtilis expresses two different thymidylate synthases: bsTS-A and bsTS-B. bsTS-A displays enhanced enzymatic and structural thermal stability uncharacteristic of most TSs. Despite the high level of TS conservation across most species, bsTS-A shares low sequence identity (<40%) with the majority of TSs from other organisms. This TS and the TSs from Lactococcus lactis and phage Phi3T-to which it is most similar-have been of interest for some time since, by structure-based sequence alignment, they appear to lack several key residues shown by mutagenesis to be essential to enzymatic function [Greene, P. J., Yu, P. L., Zhao, J., Schiffer, C. A., and Santi, D. (1994) Protein Sci. 3, 1114-6]. In addition, bsTS-A demonstrates specific activity 2-3-fold higher than TS from Lactobacillus casei or Escherichia coli. We have solved the crystal structure of this unusual TS in four crystal forms to a maximum resolution of 1.7 A. Each of these crystal forms contains either one or two noncrystallographically related dimers. Stabilization of the beta-sheet dimer interface through a dramatic architecture of buttressed internal salt bridges maintains the structural integrity of bsTS-A at elevated temperatures. Melting curves of TSs from L. casei and E. coli are compared to that of TS-A from B. subtilis and correlated with numbers of hydrogen bonds, salt bridges, and the numbers of interactions localized to the dimer interface. Analysis of this structure will shed light on the conservation of function across diversity of sequence, as well as provide insights into the thermal stabilization of a highly conserved enzyme.

Chemotherapeutic drug activation of the AP24 protease in apoptosis: requirement for caspase 3-like-proteases.

AP24 is a serine protease that is activated during TNF or UV light-induced apoptosis and stimulates DNA fragmentation in isolated nuclei. The present study determined whether apoptosis induced by chemotherapeutic drugs resulted in activation of AP24 and examined the possible relationship to caspase activity. We showed that an inhibitor of AP24, DK120, could block DNA fragmentation induced in three leukemia cell lines (U937, HL-60, and CEM) by various DNA-damaging drugs including etoposide, camptothecin, chlorambucil, and the CC1065-related drug, YW201. Etoposide-induced activation of intracellular DEVD-pNa cleaving activity and apoptosis was suppressed by low micromolar concentrations of cell-permeable inhibitors of caspase-3. Furthermore, these inhibitors also suppressed activation of AP24. In contrast, DK120 did not prevent etoposide activation of DEVD-pNa cleaving activity, nor did it prevent cleavage of poly(ADP-ribose) polymerase. AP24 isolated from apoptotic cells following treatment with etoposide activated DNA fragmentation in isolated normal nuclei and was inhibited by DK120, but not by caspase inhibitors. This evidence shows that activation of caspase 3-like proteases generates signals that contribute to the activation of AP24 which may then induce nuclear DNA fragmentation in chemotherapeutic drug-induced apoptosis.

Activation of CPP32-like proteases is not sufficient to trigger apoptosis: inhibition of apoptosis by agents that suppress activation of AP24, but not CPP32-like activity.

The 24-kD apoptotic protease (AP24) is a serine protease that is activated during apoptosis and has the capacity to activate internucleosomal DNA fragmentation in isolated nuclei. This study examined the following: (a) the functional relationship between AP24 and the CPP32-like proteases of the caspase family; and (b) whether activation of CPP32-like proteases is sufficient to commit irreversibly a cell to apoptotic death. In three different leukemia cell lines, we showed that agents that directly (carbobenzoxy-Ala-Ala-borophe (DK120) or indirectly inhibit activation of AP24 (protein kinase inhibitors, basic fibroblast growth factor, tosylphenylalaninechloromethylketone, and caspase inhibitors) protected cells from apoptosis induced by TNF or UV light. Only the caspase inhibitors, however, prevented activation of CPP32-like activity as revealed by cleavage of the synthetic substrate, DEVD-pNa, by cell cytosols, and also by in vivo cleavage of poly (ADP-ribosyl) polymerase, a known substrate of CPP32. Activation of DEVD-pNa cleaving activity without apoptosis was also demonstrated in two variants derived from the U937 monocytic leukemia in the absence of exogenous inhibitors. Cell-permeable peptide inhibitors selective for CPP32-like proteases suppressed AP24 activation and apoptotic death. These findings indicate that CPP32-like activity is one of several upstream signals required for AP24 activation. Furthermore, activation of CPP32-like proteases alone is not sufficient to commit irreversibly a cell to apoptotic death under conditions where activation of AP24 is inhibited.

Calmodulin-dependent protein kinase II mediates signal transduction in apoptosis.

The present studies describe a new function for calmodulin-dependent protein kinase II (CaM-KII) in signal transduction leading to apoptosis. Both tumor necrosis factor alpha (TNF) and UV light rapidly stimulated Ca2+-independent activity of CaM-KII in the monocytic leukemia, U937. Two mechanistically different inhibitors of CaM-KII blocked activation of CaM-KII and prevented DNA fragmentation and death. Activation of CaM-KII during apoptosis and inhibition of DNA fragmentation by the two CaM-KII inhibitors were reproduced in several other lines including KGla, HL-60, and YAC-1. However, K562, which is relatively resistant to apoptosis induced by either TNF or UV light, did not activate CaM-KII in response to these stimuli. A variant derived from U937 that is resistant to TNF- or UV light-induced apoptosis also lacked a CaM-KII response. Activation of Cam-KII was blocked by two protease inhibitors, VAD-fmk and TPCK, but not by other inhibitors of serine proteases. Both inhibitors of CaM-KII and the protease inhibitors blocked activation of AP24, a serine protease originally isolated from apoptotic cells that induces DNA fragmentation in nuclei. Our evidence supports a model in which proteolytic activity functions upstream of CaM-KII. This kinase then leads to activation of AP24, which transmits signals to the nucleus to initiate DNA fragmentation.

Cloning, expression, purification, and characterization of 2'-deoxyuridylate hydroxymethylase from phage SPO1.

2'-Deoxyuridylate hydroxymethylase (dUMP-hmase) from phage SPO1 has been cloned and expressed in Escherichia coli. In crude extracts, the enzyme represents about 25% of the soluble protein and has a higher specific activity than the most purified preparation yet reported. The enzyme was purified to homogeneity by ion-exchange and hydrophobic chromatography. The subunits of dUMP-hmase are 45 kDa by SDS-PAGE and form dimers with a molecular mass of 89.2 kDa by analytical centrifugation. In addition to the normal reaction, dUMP-hmase catalyzes the 5,10-methylene-5,6,7,8-tetrahydrofolate (CH2H4folate)-independent tritium exchange of [5-3H]dUMP for protons of water and dehalogenation of 5-bromo-2'-deoxy-uridine-5'-monophosphate; the enzyme also forms a covalent binary adduct with pyridoxal 5'-monophosphate and a covalent ternary complex with 5-fluoro-2'-deoxyuridine-5'-monophosphate and CH2H4folate. Folic acid inhibits the tritium release catalyzed by dUMP-hmase in the presence of cofactor but has no effect on the catalysis of cofactor-independent tritium exchange.

Partial restoration of activity to Lactobacillus casei thymidylate synthase following inactivation by domain deletion.

Thymidylate synthase (TS) from Lactobacillus casei has a 50 amino acid insert (residues 90-139) in the small domain that is found in only one other TS. A deletion mutant was constructed which lacked the entire insert, thereby reducing the small domain to the size found in Escherichia coli TS. This mutant did not catalyze the formation of dTMP. From the crystal structure of L. casei TS, we surmised that the loss of activity might have resulted from the exposure of residues of helices C and D, which were previously buried by the insert. To restore the local structure of helices C and D in the deletion mutants, we replaced several residues in this region by the corresponding residues found in E. coli TS. The mutant whose sequence most closely resembled that of E. coli TS carried six mutations and possessed partially restored TS activity. The mutant which had all those mutations except F87D did not catalyze any dTMP formation. The crucial role of F87D was proven in a deletion mutant which had only this change and showed greatly increased activity. All of the mutants catalyzed the debromination of BrdUMP in the absence of cofactor about as well as wild type TS. The kinetic parameters for dTMP formation of the active mutants show that the deletion has its major effect on kcat and binding of cofactor CH2H4folate, with less effect on binding of the substrate dUMP. Removal of residues 90-139 is believed to disorder helices C and D, which in turn decreases cofactor binding and catalysis.

How to predict product yield in kinetically controlled enzymatic peptide synthesis.

The published theory of kinetically controlled enzymatic peptide synthesis needed experimental verification. We carried out the measurement of the peptide yield and estimation of the key parameters alpha and beta for papain-catalyzed synthesis of Mal-L-Phe-L-Ala-LLeuNH(2) from Mal-L-Phe-L-AlaOMe and L-LeuNH(2). The experimental results demonstrate that this theory adequately describes the real process.

Protease-catalyzed peptide synthesis using inverse substrates: the synthesis of Pro-Xaa-bonds by trypsin.

Benzyloxycarbonyl-L-proline p-guanidinophenyl ester is an "inverse substrate" for trypsin; i.e., the cationic center is included in the leaving group instead of being in the acyl moiety. This substrate can be used in trypsin-catalyzed acyl-transfer reactions leading to the synthesis of Pro-Xaa peptide bonds. The reaction proceeds about 20 times slower than reaction with similar alanine-containing substrates, but the ratio between synthesis and hydrolysis is more favorable. The investigation of a series of nucleophiles led to information about the specificity of the process. Nucleophiles differing only in the P(1)'-position show an increasing acyl transfer efficiency in the order Phe < Gly < Ley < Ser < Ala < lle. C terminal elongation of the nucleophiles is of minor influence on their efficiency. The formation of an H bond between the acyl-enzyme and the nucleophile seems to play an important role in the aminolysis of the acyl-enzyme.

[Peptide synthesis catalyzed by proteases. The effect of temperature on kinetics of acyl transfer catalyzed by papain]. / Peptidnyi sintez, kataliziruemyi proteazami. Vliianie temperatury na kinetiku atsil'nogo perenosa, kataliziruemogo papainom.

The effect of temperature on the kinetics of papain-catalyzed peptide synthesis was studied. A characteristic feature of this process is that the peptide synthesis is accompanied both by the hydrolysis of the synthesized bond and by the further elongation of the peptide chain. These phenomena yield the maximum on the time dependence of the first synthetic product. A decrease in temperature is an effective way to increase the yield of this substance which reflects the increase in the nucleophil reactivity resulting from the temperature decrease. Moreover, the latter diminishes the contribution of secondary enzymatic reactions such as further hydrolysis of the reaction product and polypeptide chain elongation.

Characterization of the S'-subsite specificity of bovine pancreatic alpha-chymotrypsin via acyl transfer to added nucleophiles.

The S'-subsite specificity of bovine pancreatic alpha-chymotrypsin was investigated by acyl transfer reactions using a series of amino-acid- and peptide-derived nucleophiles. The nucleophilic efficiency covers a range of more than three orders of magnitude, reflecting the specificity of the acyl transfer process. Positively charged H-Arg-NH2 was the most efficient nucleophile of the series while peptides with free carboxyl groups show poor nucleophilic behaviour. This is explained by electrostatic interactions with the residues Asp35 and Asp64 of the enzyme. These negatively charged groups, which are localized near the appropriate S' binding sites, repel carboxylate groups of the nucleophiles. There is a good correlation between the nucleophile efficiencies found for different acyl enzymes. An investigation of a series of 14 water-soluble acyl donor esters, differing both in the P1 residue and in the number of amino acids, revealed that the nature of the acyl group affected the acyl-enzyme partitioning between water and added nucleophile in the range of one order of magnitude.

Characterization of the S'-subsite specificity of porcine pancreatic elastase.

A number of maleyl peptide p-nitrobenzyl esters have been synthesized to study elastase-catalyzed hydrolysis reactions. These new substrates were used as acyl donors to investigate the S'-subsite specificity of porcine pancreatic elastase by partitioning of the acyl enzyme between various added nucleophiles and water. The following results were obtained. 1. Porcine pancreatic elastase prefers amino acid residues with small side chains in the P'1 position. 2. The nucleophile binding is improved by a positively charged P'1 side chain, whereas a negatively charged function results in a very low binding tendency. 3. Elongation of the nucleophile to the P'2 position leads to higher aminolysis rates. 4. S' specificity is substantially influenced by the P1 residue of the acyl enzyme.

An apparatus for continuous analysis of protease-catalyzed acyl transfer reactions.

An apparatus that allows continuous analysis of protease-catalyzed acyl transfer reactions is described. Hydrolysis reaction is assayed using automatic titration. A continuous determination of amino group concentration by reaction with o-phthalaldehyde gives the rate of peptide bond formation. The apparatus allows the determination of the partition constant for the nucleophile at various nucleophile concentrations from one run.