Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate

Hen egg-white lysozyme (HEWL) was the first enzyme to have its three-dimensional structure determined by X-ray diffraction techniques(1). A catalytic mechanism, featuring a long-lived oxo-carbenium-ion intermediate, was proposed on the basis of model-building studies(2). The `Phillips' mechanism is widely held as the paradigm for the catalytic mechanism of beta -glycosidases that cleave glycosidic linkages with net retention of configuration of the anomeric centre. Studies with other retaining beta -glycosidases, however, provide strong evidence pointing to a common mechanism for these enzymes that involves a covalent glycosyl-enzyme intermediate, as previously postulated(3). Here we show, in three different cases using electrospray ionization mass spectrometry, a catalytically competent covalent glycosyl-enzyme intermediate during the catalytic cycle of HEWL. We also show the three-dimensional structure of this intermediate as determined by Xray diffraction. We formulate a general catalytic mechanism for all retaining beta -glycosidases that includes substrate distortion, formation of a covalent intermediate, and the electrophilic migration of C1 along the reaction coordinate

Ca2+ /S100 regulation of giant protein kinases

Protein phosphorylation by protein kinases plays a central regulatory role in cellular processes and these kinases are themselves tightly regulated(1). One common mechanism of regulation involves Ca2+-binding proteins (CaBP) such as calmodulin (CaM)(2). Here we report a Ca2+-effector mechanism for protein kinase activation by demonstrating the specific and >1,000-fold activation of the myosin-associated giant protein kinase twitchin by Ca2+/S100A1(2). S100A1(2) is a member of a large CaBP family that is implicated in various cellular processes, including cell growth, differentiation and motility, but whose molecular actions are largely unknown(3). The S100A1(2)-binding site is a part of the autoregulatory sequence positioned in the active site that is responsible for intrasteric autoinhibition of twitchin kinase; the mechanism of autoinhibition based on the crystal structures of two twitchin kinase fragments is described elsewhere(4). Ca2+/S100 represents a likely physiological activator for the entire family of giant protein kinases involved in muscle contractions and cytoskeletal structure(2,5-9)

CRYSTALLOGRAPHIC ANALYSIS OF THE CATALYTIC MECHANISM OF HALOALKANE DEHALOGENASE

Crystal structures of haloalkane dehalogenase were determined in the presence of the substrate 1,2-dichloroethane. At pH 5 and 4-degrees-C, substrate is bound in the active site without being converted; warming to room temperature causes the substrate's carbon-chlorine bond to be broken, producing a chloride ion with concomitant alkylation of the active-site residue Asp124. At pH 6 and room temperature the alkylated enzyme is hydrolysed by a water molecule activated by the His289-Asp260 pair in the active site. These results show that catalysis by the dehalogenase proceeds by a two-step mechanism involving an ester intermediate covalently bound at Asp124

Doughnut-shaped structure of a bacterial muramidase revealed by X-ray crystallography

The integrity of the bacterial cell wall depends on the balanced action of several peptidoglycan (murein) synthesizing and degrading enzymes. Penicillin inhibits the enzymes responsible for peptide crosslinks in the peptidoglycan polymer. Enzymes that act solely on the glycosidic bonds are insensitive to this antibiotic, thus offering a target for the design of antibiotics distinct from the β-lactams. Here we report the X-ray structure of the periplasmic soluble lytic transglycosylase (SLT; Mr 70,000) from Escherichia coli. This unique bacterial exomuramidase cleaves the β-1,4-glycosidic bonds of peptidoglycan to produce small 1,6-anhydromuropeptides.The structure of SLT reveals a 'superhelical' ring of α-helices with a separate domain on top which resembles the fold of lysozyme. Site-directed mutagenesis and a crystallographic inhibitor-binding study confirmed that the lysozyme-like domain contains the active site of SLT.