Mutagenesis studies

En me Mechanism. Staphylococcal nuclease (SNase) accelerates the hydrolysis of phosphodiester bonds in nucleic acids (qv) some 10 -fold over the uncatalyzed rate (r93 and references therein). Mutagenesis studies in which Glu43 has been replaced by Asp or Gin have shown Glu to be important for high catalytic activity. The enzyme mechanism is thought to involve base catalysis in which Glu43 acts as a general base and activates a water molecule that attacks the phosphodiester backbone of DNA. To study this mechanistic possibiUty further, Glu was replaced by two unnatural amino acids. 

Q Zeng, ET Smith, DM Kurtz, RA Scott. Protein determinants of metal site reduction potentials. Site directed mutagenesis studies of Clostridium pasteurianum laibredoxin. Inorg Chim Acta 242 245-251, 1996. 

A unique feature of the interaction of the hormone and PLR is at the beginning of the F-G loop in the C-terminal domain. In HGR the sequence is Arg-Asn-Ser whereas in PLR it is Asp-His-deletion. This loop interacts with His 18 and Glu 174 of the hormone. In PLR the orientation of this loop is such that the Asp and His residues, in combination with His and Glu from the hormone, form a strong binding site for a zinc atom that links the hormone and the receptor (Figure 13.23b). The presence of zinc increases the affinity of the hormone for the receptor in vitro by a factor of 10,000. As shown by mutagenesis studies His 18 and Glu 174 of the hormone are important for the tight binding to PRL but not to GHR. 

Branchini, B. R., et al. (2003). A mutagenesis study of the putative luciferin binding site residues of firefly luciferase. Biochemistry 42 10429-10436. 

Upon activation by CXCLl2, CXCR4 is quickly phosphorylated and internalized. Several residues in its C-terminus tail have been identified as potential phosphorylation sites by truncation and mutagenesis studies as reviewed in Busillo and Benovic (2007). Removal of 45 amino acid residues from the C-terminal of CXCR4 led to elimination of CXCL12-induced phoshorylation, enhanced receptor activity... 

KAO Y c, ZHOU c, SHERMAN M, LAUGHTON 0 A, CHEN s (1998) Molecular basis of the inhibition of human aromatase (oestrogen synthetase) by flavone and isoflavone phytoestrogens a site-directed mutagenesis study. Environ Health Perspect. 106 85-92. 

Williams G, Borkakoti N, Bottomley GA, et al. Mutagenesis studies of interleukin-8. Identification of a second epitope involved in receptor binding. J Biol Chem... 

Mayo KH, Ilyina E, Roongta V, et al. Heparin binding to platelet factor-4. An NMR and site-directed mutagenesis study arginine residues are crucial for binding. Biochem J 1995 312 357-65. 

While the ddNs and ANPs must be converted intracellularly to their 5 -triphosphates (ddNTPs) or diphosphate derivatives before they can interact as competitive inhibitors/alternate substrates with regard to the natural substrates (dNTPs), the NNRTIs do not need any metabolic conversion to interact, noncompetitively with respect to the dNTPs, at an allosteric, non-substrate binding site of the HIV-1 RT. Through the analysis of NNRTI-resistant mutants, combined with site-directed mutagenesis studies, it has become increasingly clear which amino acid residues are involved in the interaction of the NNRTIs with HIV-1 RT, and, since the conformation of the HIV-1 RT has been resolved at 3.0 A resolution [73], it is now possible to visualize the binding site of the NNRTIs [74],... 

---