@article {2378,
	title = {NMR Spectroscopic Characterization of the Sialyltransferase CstII from Campylobacter jejuni: Histidine 188 Is the General Base},
	journal = {Biochemistry},
	volume = {48},
	number = {47},
	year = {2009},
	note = {ISI Document Delivery No.: 521UVTimes Cited: 2Cited Reference Count: 55Chan, Patrick H. W. Lairson, Luke L. Lee, Ho Jun Wakarchuk, Warren W. Strynadka, Natalie C. J. Withers, Stephen G. McIntosh, Lawrence P.},
	month = {Dec},
	pages = {11220-11230},
	type = {Article},
	abstract = {Cell surface glycans are often terminated by sialic acid, which is incorporated onto sugar acceptors by sialyltransferases. The crystal structure of the GT family 42 Campylobacter jejuni alpha-2,3/2,8-sialyltransferase (CstII) provides key insights into the sialyl-transfer mechanism, Including tentative identification of His 188 as the catalytic base. In support of this hypothesis, the CstII-H188A mutant is able to catalyze sialyl transfer from CMP-Neu5Ac to added anions such as azide and formate but not to its natural sugar acceptor lactose. Complementing this work, NMR spectroscopy was used to investigate the Structure and dynamics of CstII and to measure the intrinsic pK(a) value of His 188 for comparison with the pK(a) determined from the pH-dependent k(cat)/K-M of the enzyme. By systematically introducing point mutations at the subunit interfaces, two active monomeric variants, CstII-F121D and CstII-Y125Q, were obtained and characterized. In contrast to the wild-type tetramer, the monomeric CstII variants yielded good quality H-1/N-15-HSQC and H-1/C-13-methyl-TROSY NMR spectra. However, the absence of signals from approximately one-half of the amides in the H-1/N-15-HSQC spectra of both monomeric forms suggests that the enzyme undergoes substantial conformational exchange on a millisecond to microsecond time scale. The histidine pK(a) values of CstII-F121D in its apo form were measured by monitoring the pH-dependent chemical shifts of [C-13(epsilon l)]histidine, biosynthetically incorporated into the otherwise uniformly deuterated protein. Consistent with its proposed catalytic role, the site-specific pK(a) value similar to 6.6 of His 188 matches the apparent pK(a) value similar to 6.5 governing the pH dependence of k(cat)/K-M for CstII toward CMP-Neu5Ac in the presence of saturating acceptor substrate.},
	keywords = {20S PROTEASOME, CHEMICAL RESCUE, CRYSTAL-STRUCTURE, GANGLIOSIDE MIMICS, LABELING STRATEGY, MILLER-FISHER-SYNDROMES, SIALIC-ACID, STRUCTURAL-ANALYSIS, SUBSTRATE-ANALOG, TRANS-SIALIDASE},
	isbn = {0006-2960},
	url = {<Go to ISI>://000271950900011},
	author = {Chan, P. H. W. and Lairson, L. L. and Lee, H. J. and Wakarchuk, W. W. and Strynadka, N. C. J. and Withers, S. G. and McIntosh, L. P.}
}
@article {2635,
	title = {Probing General Acid Catalysis in the Hammerhead Ribozyme},
	journal = {Journal of the American Chemical Society},
	volume = {131},
	number = {3},
	year = {2009},
	note = {ISI Document Delivery No.: 427PQTimes Cited: 5Cited Reference Count: 58Thomas, Jason M. Perrin, David M.},
	month = {Jan},
	pages = {1135-1143},
	type = {Article},
	abstract = {Recent crystallographic and computational studies have provided fresh insights into the catalytic mechanism of the RNA-cleaving hammerhead ribozyme. Based on these findings, specific ribozyme functional groups have been hypothesized to act directly as the general acid and base catalysts, although the catalytic role of divalent metal cations (M2+) remains uncertain: We now report a functional characterization of the general acid catalysis mechanism and the role of an M2+ cofactor therein, for the S. mansoni hammerhead (an "extended" hammerhead ribozyme). We have compared hammerhead cleavage of substrates with natural (ribo-phosphodiester) versus bridging-5{\textquoteright}-phosphorothioate scissile linkages, in the contexts of active site mutations and M2+ substitution. Cleavage of the natural substrate is inhibited by modification of the G8 2{\textquoteright}-OH ribozyme residue and depends strongly upon the presence and identity of an M2+ cofactor; in contrast, cleavage of the bridging-phosphorothioate substrate is conspicuously insensitive to any of these factors. These results imply that (1) both an M2+ cofactor and the G8 2{\textquoteright}-OH play crucial roles in hammerhead general acid catalysis and (2) the M2+ cofactor does not contribute to general acid catalysis via Lewis acid stabilization of the leaving group. General acid pK(a) perturbation was also demonstrated for both M2+ substitution and G8 2{\textquoteright}-OH modification, which suggests transition state M2+ coordination of the G8 2{\textquoteright}-OH, to lower its pK(a) and improve its ability to transfer a proton to the leaving group. We also report a simple method for synthesizing radiolabeled bridging-5{\textquoteright}-phosphorothioate substrates.},
	keywords = {2{\textquoteright}-HYDROXYL GROUP, ACTIVE-SITE, BASE CATALYSIS, CATIONS, MEDIATED CLEAVAGE, METAL-ION, MONOVALENT, P-31 NMR, PHOSPHOLIPASE-C, RIBONUCLEASE-A, SUBSTRATE-ANALOG},
	isbn = {0002-7863},
	url = {<Go to ISI>://000264791600054},
	author = {Thomas, J. M. and Perrin,David M.}
}
