Substrate (chemistry)

About: Substrate (chemistry) is a research topic. Over the lifetime, 35902 publications have been published within this topic receiving 740722 citations. The topic is also known as: enzyme substrate.

Structural basis for substrate loading in bacterial RNA polymerase

Abstract: The mechanism of substrate loading in multisubunit RNA polymerase is crucial for understanding the general principles of transcription yet remains hotly debated Here we report the 30-A resolution structures of the Thermus thermophilus elongation complex (EC) with a non-hydrolysable substrate analogue, adenosine-5′-[(α,β)-methyleno]-triphosphate (AMPcPP), and with AMPcPP plus the inhibitor streptolydigin In the EC/AMPcPP structure, the substrate binds to the active (‘insertion’) site closed through refolding of the trigger loop (TL) into two α-helices In contrast, the EC/AMPcPP/streptolydigin structure reveals an inactive (‘preinsertion’) substrate configuration stabilized by streptolydigin-induced displacement of the TL Our structural and biochemical data suggest that refolding of the TL is vital for catalysis and have three main implications First, despite differences in the details, the two-step preinsertion/insertion mechanism of substrate loading may be universal for all RNA polymerases Second, freezing of the preinsertion state is an attractive target for the design of novel antibiotics Last, the TL emerges as a prominent target whose refolding can be modulated by regulatory factors Two complementary papers this week focus on the structure and function of bacterial RNA polymerase In the first, the enzyme is bound to the DNA template and RNA product, to give a close-up of the transcription elongation complex The structure reveals details of the DNA-to-RNA transcription process, vital to all living cells In the second paper, the RNA polymerase elongation complex is pictured bound to various substrate analogues and to an antibiotic, revealing the mechanism of substrate loading and antibiotic inhibition Comparisons between the structures of human and bacteria RNA polymerase should aid in drug design: RNA polymerase is a target of antibiotics, including rifamycin and its derivatives Crystal structures of bacterial RNA polymerase elongation complexes bound to NTP substrate analogues with an antibiotic, revealing the mechanism of substrate loading and antibiotic inhibition

Redesigning enzyme structure by site-directed mutagenesis: tyrosyl tRNA synthetase and ATP binding

Abstract: We describe here a general method for systematically replacing amino acids in an enzyme. This allows analysis of their molecular roles in substrate binding or catalysis and could eventually lead to the engineering of new enzymatic activities. The gene encoding the enzyme is first cloned into a vector from which the enzyme is expressed and is then mutated in vitro to change a particular nucleotide and hence the amino acid sequence of the enzyme. We have cloned the gene for the tyrosyl tRNA synthetase of Bacillus stearothermophilus into a vector derived from the single-stranded bacteriophage M13 to facilitate mutagenesis with mismatched synthetic oligodeoxynucleotide primers. From the recombinant M13 clone we have obtained high levels of the enzyme (∼50% of soluble protein) expressed in the Escherichia coli host and have converted cysteine (Cys35) at the enzyme's active site to serine. This leads to a reduction in enzymatic activity that is largely attributable to a lower Km for ATP.

Binding of chloromethyl ketone substrate analogs to crystalline papain

Abstract: Papain (EC 3.4.22.2) is a proteolytic enzyme, the three-dimensional structure of which has been determined by x-ray diffraction at 2.8 A resolution (Drenth, J., Jansonius, J.N., Koekoek, R., Swen, H. M., and Wothers, B.G. (1968), Nature (London) 218, 929-932). The active site is a groove on the molecular surface in which the essential sulfhydryl group of cysteine-25 is situated next to the imidazole ring of histidine-159. The main object of this study was to determine by the difference-Fourier technique the binding mode for the substrate in the groove in order to explain the substrate specificity of the enzyme (P2 should have a hydrophobic side chain (Berger and Schechter, 1970) and to contribute to an elucidation of the catalytic mechanism. To this end, three chloromethyl ketone substrate analogues were reacted with the enzyme by covalent attachment to the sulfur atom of cysteine-25. The products crystallized isomorphously with the parent structure that is not the native, active enzyme but a mixture of oxidized papain (probably papain-SO2-) and papain with an extra cysteine attached to cysteine-25. Although this made the interpretation of the difference electron density maps less easy, it provided us with a clear picture of the way in which the acyl part of the substrate binds in the active site groove. The carbonyl oxygen of the P1 residue is near two potential hydrogen-bond donating groups, the backbone NH of cysteine-25 and the NH2 of glutamine-19. Valine residues 133 and 157 are responsible for the preference of papain in its substrate splitting. By removing the methylene group that covalently attaches the inhibitor molecules to the sulfur atom of cysteine-25 we obtained acceptable models for the acyl-enzyme structure and for the tetrahedral intermediate. The carbonyl oxygen of the P1 residue, carrying a formal negative charge in the tetrahedral intermediate, is stabilized by formation of two hydrogen bonds with the backbone NH of cysteine-25 and the NH2 group of glutamine-19. This situation resembles that suggested for the proteolytic serine enzymes (Henderson, R., Wright, C. S., Hess, G. P., and Blow, D. M. (1971), Cold Spring Harbor Symp. Quant. Biol. 36, 63-70; Robertus, J. D., Kraut, J., Alden, R. A., and Birktoft, J. J. (1972b), Biochemistry 11, 4293-4303). The nitrogen atom of the scissile peptide bond was found close to the imidazole ring of histidine-159, suggesting a role for this ring in protonating the N atom of the leaving group (Lowe, 1970). This proton transfer would be facilitated by a 30 degrees rotation of the ring around the C beta-Cgamma bond from an in-plane position with the sulfur atom to an in-plane position with the N atom. The possibility of this rotation is derived from a difference electron-density map for fully oxidizied papain vs. the parent protein.