Crystals of bovine trypsin were acylated at the reactive residue, serine 195, to form the transiently stable p-guanidinobenzoate. Hydrolysis of this species was triggered in the crystals by a jump in pH. The hydrolysis was monitored by three-dimensional Laue crystallography, resulting in three x-ray diffraction structures, all from the same crystal and each representing approximately 5 seconds of x-ray exposure. The structures were analyzed at a nominal resolution of 1.8 angstroms and were of sufficient quality to reproduce subtle features in the electron-density maps for each of the structures. Comparison of the structures before and after the pH jump reveals that a water molecule has positioned itself to attack the acyl group in the initial step of the hydrolysis of this transient intermediate.

The three-dimensional structures of the transforming region of the product of the EJ/T24 human bladder oncogene and of the c-Ha ras8-gene product have been calculated by using conformational energy calculations. These two genes, representing a transforming oncogene and its normal cellular homologue, encode 21,000-dalton peptides that differ by one amino acid at position 12. We therefore examined the energetically allowed conformations of the hydrophobic decapeptide surrounding this substitution site. The calculations show that the most favorable form of the c-Ha ras-l gene product exists when glycine-12 is in a left-handed bend conformation. No other amino acid can adopt this conformation and thus the bladder oncogene peptide containing valine at position 12 has a markedly different three-dimensional structure. A simple model is proposed to account for the consequences of a position 12 mutation.by restriction mapping (12)(13)(14). In our routine DNA transfection assays, however, the cloned oncogene has a transforming potency that is several orders of magnitude higher than that of cloned c-Ha ras-1. By chimeric reconstruction, this difference in transforming potency was localized to a 350-nucleotide sequence in the 5' end of the p21 coding sequences. The sequences of these regions of DNA have been determined (15)(16)(17), and the difference in transforming potential was pinpointed to a single-base change-from guanosine to thymidine. This mutation results in substitution of valine for glycine at residue 12 of the predicted amino acid sequence of p21 and occurs in the middle of a hydrophobic sequence. The sequence for the first 20 amino acid residues for the normal (nontransforming) protein is Harvey murine sarcoma virus (Ha-MuSV) was originally isolated from a tumor-bearing rat that had been inoculated with Moloney murine leukemia virus (1). The resultant retrovirus induces solid tumors and erythroleukemia in susceptible mice (2), and the naked viral DNA is capable of transforming NIH 3T3 cells after transfection (3). The region of the viral DNA responsible for the transforming activity has been localized to a one-kilobase segment of nucleic acid derived from the original host rat. A 21,000-dalton protein (p21) is encoded by this region of the viral DNA (4-7). Sequences homologous to the Ha-MuSV transforming region have been molecularly cloned from normal rat DNA (8). Moreover, homology between these sequences and DNA from other species has allowed the identification and molecular cloning from normal human DNA of genes related to the oncogene of Ha-MuSV (9). These cellular homologues (conc or more specifically in this case c-Ha ras) encode a 21,000-dalton protein immunologically and biochemically related to viral p21 (10).Through DNA transfer techniques, oncogenic sequences have been identified in several human tumors (11). The tumor oncogenes found in T24 and EJ human bladder carcinoma cell lines have been shown to be related to the c-Ha ras gene (12)(13)(14). The normal cellular counterpart of a tumor o...

Crystal structures of two forms of ribonuclease A with deoxynucleosides covalently bound to respectively His 12 and His 119 have been solved. One form, T-H12-RNase, has a deoxythymidine bound to N epsilon 2 of His 12, while the other one, U-H119-RNase, has a deoxyuridine bound to N delta 1 of His 119. The two crystal forms are nearly isomorphous, with two molecules in the asymmetric unit. However, the modified ribonucleases differ both in their enzymatic activities and in the conformation of the catalytic site and of the deoxynucleoside-histidine moiety. T-H12-RNase is characterized by complete loss of enzymatic activity; in this form the deoxynucleoside completely blocks the catalytic site and forms intramolecular contacts with residues associated with both the B1 and B2 sites. U-H119-RNase retains 1% of the activity of the unmodified enzyme, and in this form His 119 adopts a different orientation, corresponding to the alternate conformation reported for this residue; the deoxynucleoside-histidine moiety points out of the active site and does not form any contacts with the rest of the protein, thus allowing partial access to the catalytic site. On the basis of these structures, we propose possible mechanisms for the reactions of bromoacetamido nucleosides with ribonuclease A.

The binding of cancer cells to the basement membrane glycoprotein laminin appears to be a critical step in the metastatic process. This binding can be inhibited competitively by a specific pentapeptide sequence (Tyr-Ile-Gly-Ser-Arg) of the laminin B1 chain, and this peptide can prevent metastasis formation in vivo. However, other similar pentapeptide sequences (e.g., Tyr-Ile-Gly-Ser-Glu) have been found to be much less active in metastasis inhibition, raising the possibility that such amino acid substitutions produce structural changes responsible for altering binding to the laminin receptor. In this study, conformational energy analysis has been used to determine the three-dimensional structures of these peptides. The results indicate that the substitution of Glu for the terminal Arg produces a significant conformational change in the peptide backbone at the middle Gly residue. These results have important implications for the design of drugs that may be useful in preventing metastasis formation and tumor spread.

Purified matrix vesicle alkaline phosphatase from bovine fetal epiphyseal cartilage hydrolyzes a variety of phosphate esters as well as ATP and PP~. Optimal activities for p-nitrophenyl phosphate, ATP, and PP~ are found at pH 10.5, 10.0, and 8.5, respectively. The latter two substrates exhibit substrate inhibition at high concentrations, p-Nitrophenyl phosphate demonstrates decreasing pH optima with decreasing substrate concentration. Heat inactivation studies indicate that both phosphorohydrolytic and pyrophosphorolytic cleavage occur at the same site on the enzyme. Mg'-'* and Hg"* ions inhibit the p-nitrophenyl phosphatase activity at pH 10.5 while Mn 2. ions show no effect. Pi, levamisole, CN-, Zn 2 § Ca ~ § ions, and L-phenylalanine are reversible inhibitors of the phosphomonoesterase activity. P~ is a linear noncompetitive inhibitor with a K~ of 8.0 mM. Levamisole and L-phenylalanine are uncompetitive inhibitors with inhibition constants of 0.02 and 39.4 mM, respectively. Ca "~ ions inhibit noncompetitively with a K~ of 9.3 raM. Zn 2+ ion is a potent noncompetitive inhibitor with an inhibition constant of 0.026 mM. The enzyme is inhibited irreversibly by Be ~ ion, EDTA, EGTA, ethane-l-hydroxydiphosphonate, dichloromelhanediphosphohate, L-cysteine. and N-ethylmaleimide. NaCI, KCI. and NaeSO4 at 0.5-1.0 M inhibit the enzyme.At pH 8.5, the cleavage of PP~ by the matrix vesicle enzyme is inhibited by Mg '+ and Ca'-'~ ions at concentrations greater than 0.5 raM. Mg 2. ions in the range of 0.1-4 mM stimulate the matrix vesicle ATPase whereas higher concentrations produce inhibition. Ca'-' ~ ion does not affect the ATPase activity between 0.1 and 10 mM at either pH 7.5 or 10.0, Send ~ffl)t'int reque.~ts to Robert P. Carx} at the above address. ~Present address:

No abstract

Using conformational energy calculations, we previously predicted that there are two distinct binding modes for hexasaccharide substrates of hen egg white lysozyme (HEWL), a "left-sided" binding mode and a "right-sided" one. The former involves such residues as Arg-45, Asn-46, and Thr-47, while the latter involves such residues as Asn-113 and Arg-114. The left-sided binding mode was predicted to predominate for (GlcNAc)6. We now present two lines of experimental evidence that indicate that left-sided binding occurs for this substrate. First, we show that ring-necked pheasant lysozyme (RNPL), in which Lys and His replace Asn and Arg at positions 113 and 114, respectively, has the same affinity for (GlcNAc)6 as does HEWL, indicating that the "right" side is not involved in equilibrium binding to the substrate. Second, we show that a monoclonal antibody, HyHEL-5, which binds specifically to an epitope including residues Arg-45, Asn-46, Thr-47, Asp-48, and Arg-68 on the far "left" side of HEWL, is competitively displaced by (GlcNAc)5 and (GlcNAc)6 but not by GlcNAc, (GlcNAc)2, or (GlcNAc)4. Only the former two substrates can bind in site F in the lower active site. Since these two substrates are the only ones that competitively displace HyHEL-5, our results suggest that the terminal saccharide residues of these substrates bind to the left side of the active site cleft, as predicted from theory.

Four new bromoacetamido pyrimidine nucleosides have been synthesized and are affinity labels for the active site of bovine pancreatic ribonuclease A (RNase A). All bind reversibly to the enzyme and react covalently with it, resulting in inactivation. The binding constants Kb and the first-order decomposition rate constants k3 have been determined for each derivative. They are the following: 3'-(bromoacetamido)-3'-deoxyuridine, Kb = 0.062 M, k3 = 3.3 X 10(-4) s-1; 2'-(bromoacetamido)-2'-deoxyxylofuranosyluracil, Kb = 0.18 M, k3 = 1700 X 10(-4) s-1; 3'-(bromoacetamido)-3'-deoxyarabinofuranosyluracil, Kb = 0.038 M, k3 = 6.6 X 10(-4) s-1; and 3'-(bromoacetamido)-3'-deoxythymidine, Kb = 0.094 M, k3 = 2.7 X 10(-4) s-1. 3'-(Bromoacetamido)-3'-deoxyuridine reacts exclusively with the histidine-119 residue, giving 70% of a monoalkylated product substituted at N-1, 14% of a monoalkylated derivative substituted at N-3, and 16% of a dialkylated species substituted at both N-1 and N-3. Both 2'-(bromoacetamido)-2'-deoxyxylofuranosyluracil and 3'-(bromoacetamido)-3'-deoxyarabinofuranosyluracil react with absolute specificity at N-3 of the histidine-12 residue. 3'-(Bromoacetamido)-3'-deoxythymidine alkylates histidines-12 and -119. The major product formed in 57% yield is substituted at N-3 of histidine-12. A monoalkylated derivative, 8% yield, is substituted at N-1 of histidine-119. A disubstituted species is formed in 14% yield and is alkylated at both N-3 of histidine-12 and N-1 of histidine-119. A specific interaction of the "down" 2'-OH group, unique to 3'-(bromoacetamido)-3'-deoxyuridine, serves to orient the 3'-bromoacetamido residue close to the imidazole ring of histidine-119. The 2'-OH group of 3',5'-dinucleoside phosphate substrates may serve a similar role in the catalytic mechanism, allowing histidine-119 to protonate the leaving group in the transphosphorylation step. (Bromoacetamido)nucleosides are bound in the active site of RNase A in a variety of distinct conformations which are responsible for the different specificities and alkylation rates.