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Biomedical subjects

J Kraut

Publications and source records attributed to J Kraut.

At least 109 records · Page 6Linked to original sources
PubMed1977

Dihydrofolate reductase: x-ray structure of the binary complex with methotrexate.

A central eight-stranded beta-pleated sheet is the main feature of the polypeptide backbone folding in dihydrofolate reductase. The innermost four strands and two bridging helices are geometrically similar to but are connected in a different way from those in the dinucleotide binding domains found in nicotinamide-adenine dinucleotide-linked dehydrogenases. Methotrexate is bound in a 15-angstrom-deep cavity with the pteridine ring buried in a primarily hydrophobic pocket, although a strong interaction occurs between the side chain of aspartic acid 27 and N(1), N(8), and the 2-amino group of methotrexate.

Binding Sites↗
PubMed1976

A detailed structural comparison between the charge relay system in chymotrypsinogen and in alpha-chymotrypsin.

An improved 2.5-A electron density map of chymotrypsinogen was calculated by incorporating heavy-atom anomalous scattering effects and a new model of the molecule was constructed. Phases from x-ray structure factors (R = 0.43) computed from this model were then used in the calculation of another electron density map against which the model was further refined. The catalytic Ser-195 side chain in the new model is in the "down" or "acyl" orientation and its Ogamma atom is in position to form a normal hydrogen bond with Nepsilon2 of His-57. In contrast, the corresponding hydrogen bond in alpha-chymotrypsin (Birktoft, J.J., and Blow, D.M. (1972), J.Mol. Biol. 68, 187) is severely distorted, probably as a consequence of a 1.5-A shift in the relative positions of the two cylindrical folding domains composing most of the molecule. We suggest that this activiation induced distortion of the charge-relay, hydrogen-bonding system plays an important role in the genesis of enzymic activity, in accord with an earlier proposal by Wang concerning the role of bent hydrogen bonds in enzyme catalysis (Wang, J.J. (1970), Proc. Natl. Acad. Sci. U.S.A. 66, 874).

Chymotrypsin↗
PubMed1976

Polypeptide halomethyl ketones bind to serine proteases as analogs of the tetrahedral intermediate. X-ray crystallographic comparison of lysine- and phenylalanine-polypeptide chloromethyl ketone-inhibited subtilisin.

1. A detailed study of cytochrome C oxidse activity with Keilin-Hartree particles and purified beef heart enzyme, at low ionic strength and low cytochrome C concentrations, showed biphasic kinetics with apparent Km1 = 5 x 10(-8) M, and apparent Km2 = 0.35 to 1.0 x 10(-6) M. Direct binding studies with purified oxidase, phospholipid-containing as well as phospholipid-depleted, demonstrated two sites of interaction of cytochrome c with the enzyme, with KD2 less than or equal to 10(-7) M, and KD2 = 10(-6) M. 2...

Amino Acid Sequence↗
PubMed1975

X-ray crystallographic study of boronic acid adducts with subtilisin BPN' (Novo). A model for the catalytic transition state.

We have studied the structures of adducts formed between subtilisin BPN' and both benzeneboronic acid and 2-phenylethaneboronic acid by x-ray diffraction techniques. Electron density and difference maps at 2.5 A resolution were computed with phases calculated from a partially refined structure of the native enzyme (R = 0.23 at 2.0 A). Both adducts contain a covalent bond between Ogamma of the catalytic Ser-221 and the inhibitor boron atom. The boron atom is coordinated tetrahedrally, with one of the two additional boronic acid oxygen atoms lying in the "oxyanion hole" and the other at the leaving group site identified in previous studies (ROBERTUS, J.D., Kraut, J. ALDEN, R.A., and BIRKTOFT, J.J. (1972) Biochemistry 11, 4293-4303). Moreover, the previously postulated structure of the tetrahedral intermediate for substrate hydrolysis is isosteric with these boronic acid adducts, which can therefore be considered good models for the transition state complex (KOEHLER, K.K., and LIENHARD, G.E. (1972) Biochemistry 10, 2477-2483). These observations further support the suggestion that an important contribution to stabilization of this transition state complex, relative to both the Michaelis complex and the acyl intermediate, occurs as a consequence of hydrogen bond donation to the substrate carbonyl oxygen atom from the side chain amido group of Asn-155 and from the backbone amido group of Ser-221.

Binding Sites↗
PubMed1975

Crystallographic structure refinement of Chromatium high potential iron protein at two Angstroms resolution.

The structure of Chromatium high potential iron protein (HiPIP) has been refined by semiautomatic Fo-Fc (observed minus calculated structure amplitude Fourier methods to a convential R index, R=sum of the absolute value of Fo-Fc divided by the sum of Fo, of 24.7% for a model in which bond distances and angles are constrained to standard values. Bond length and angle constraints were applied only intermittenly during the computations. At a late stage of the refinement, atomic parameters for only the Fe4S4 cluster plus the 4 associated cystein S-gamma atoms were adjusted by least squares methods and kept fixed during the rest of the refinement. The refined model consists of 625 of the 632 nonhydrogen atoms in the protein plus 75 water molecules. Seven side chain atoms could not be located in the final electron density map. A computer program rather than visual inspection was used wherever possible in the refinement: for locating water molecules, for removing water molecules that too closely approach other atoms, for deleting atoms that lay in regions of low electron density, and for evaluating the progress of refinement. Fo-Fc Fourier refinement is sufficiently economical to be applied routinely in protein crystal structure determinations. The complete HiPIP refinement required approximately 12 hours of CDC 3600 computer time and cost less than $3000 starting from a "trial structure," based upon multipe isomorphoous replacement phases, which gave an R of 43%...

Bacterial Proteins↗
PubMed1974

A proposed model for interaction of polypeptides with RNA.

Pairs of antiparallel beta polypeptide-chain segments in known protein structures are usually observed to form right-handed double helixes with helix parameters in the same range as those of nucleic acids. We have constructed a model containing only standard bond lengths, bond angles, and dihedral angles in which such a polypeptide double helix fits precisely into the minor groove of an RNA double helix with identical helix parameters. The geometry of the RNA portion is essentially a hybrid between those of the A and A' forms. Hydrogen bonds can be made between the ribose 2'-hydroxyls and polypeptide carbonyl oxygens. Since such precise complementarity between the stable conformations of RNA and polypeptides is unlikely to be merely coincidental, we propose that it played a fundamental role in the initiation of precellular evolution. Specificially, we propose that the two double-helical structures are mutually catalytic for assembly of one another from activated precursors in the prebiotic soup, and moreover that they provide some degree of genetic coding.

Biological Evolution↗