Chemical and mutagenic modification combined with X-ray crystallography has been used to probe the ascorbate binding site in ascorbate peroxidase (APX). Chemical modification of the single Cys residue in APX with Ellman's reagent (DTNB) blocks the ability of APX to oxidize ascorbate but not other small aromatic phenolic substrates. DTNB-modified APX (APX-TNB) exhibits only 1.3% wild-type activity when ascorbate is used as the substrate but full activity when aromatic substrates, guaiacol or pyrogallol, are used. Stopped-flow studies show that APX-TNB reacts normally with peroxide to give compound I but that the rates of reduction of both compounds I and II by ascorbate are dramatically slowed. Conversion of Cys32 to Ser leads to approximately 70% drop in ascorbate peroxidase activity with no effect on guaiacol peroxidase activity. These results indicate that uncharged aromatic substrates and the anionic ascorbate molecule interact with different sites on APX. The 2.0 A X-ray crystal structure of APX-TNB shows clear electron density for the TNB group covalently attached to Cys32 in all four molecules of the asymmetric unit, indicating complete and specific modification. It appears that the ascorbate site is blocked by DTNB modification which is well removed from the exposed delta-heme edge where aromatic substrates are thought to bind. This is the first experimental evidence indicating that ascorbate oxidation does not occur at the exposed heme edge but at an alternate binding site in the vicinity of Cys32 near Arg172 and the heme propionates.

Complete cellulose degradation is the first step in the use of biomass as a source of renewable energy. To this end, the engineering of novel cellulase activity, the activity responsible for the hydrolysis of the ␤-1,4-glycosidic bonds in cellulose, is a topic of great interest. The high-resolution X-ray crystal structure of a multidomain endoglucanase from Clostridium cellulolyticum has been determined at a 1.6-Å resolution. The endoglucanase, Cel9G, is comprised of a family 9 catalytic domain attached to a family III c cellulose-binding domain. The two domains together form a flat platform onto which crystalline cellulose is suggested to bind and be fed into the active-site cleft for endolytic hydrolysis. To further dissect the structural basis of cellulose binding and hydrolysis, the structures of Cel9G in the presence of cellobiose, cellotriose, and a DP-10 thio-oligosaccharide inhibitor were resolved at resolutions of 1.7, 1.8, and 1.9 Å, respectively.Cellulases catalyze the hydrolysis of the ␤-1,4-glycosidic linkages in cellulose, the most abundant biopolymer. Varieties of microorganisms secrete these enzymes either individually or associated in a macromolecular complex referred to as the cellulosome (23). These different cellulases, acting synergistically, catalyze the complete hydrolysis of cellulose to glucose, which, under anaerobic conditions, is a highly fermentable fuel product. The ecological and economic advantages of plant biomass conversion have spawned considerable interest in the study and eventual use of recombinant cellulolytic complexes.Clostridium cellulolyticum is an anaerobic, mesophilic, soil bacterium that is able to grow on cellulose as its sole carbon source. C. cellulolyticum secretes its cellulases in the form of a cellulosomal complex that has been extensively studied biochemically and genetically (2,14). One of these cellulases, Cel9G, is a multidomain endoglucanase capable of hydrolyzing crystalline cellulose at an appreciable rate (13). The multiple domains of this cellulase include a catalytic domain (CD) responsible for the binding and hydrolysis of cellulose, a cellulose-binding domain (CBD) responsible for the attachment of the enzyme to a cellulose chain, and the dockerin domain, which is responsible for the attachment of the cellulase to the cellulosome (35). Interestingly, in the absence of cellulosomal interactions, the dockerin domain is autolyzed in a matter of a couple of weeks at 4°C. No true cellulolytic activity is attributed to the CBD and yet, in its absence, the CD of Cel9G is inactive toward carboxymethyl cellulose, Avicel, or phosphoric acid swollen cellulose (PASC), implying a possible role in catalysis (13). In addition, when expressed as a fusion protein fused to glutathione S-transferase, the CBD alone cannot bind to cellulosic substrates.Based on sequence alignment studies, the CD of Cel9G belongs to family 9 of the glycoside hydrolase enzymes and uses a single displacement hydrolytic mechanism with net inversion of the substrate's anomeric C1 carbon. Th...

Recombinant pea cytosolic ascorbate peroxidase (APX) has been characterized by resonance Raman (RR) and electronic absorption spectroscopies. The ferric and ferrous forms together with the complexes with fluoride and imidazole have been studied and compared with the corresponding spectra of cytochrome c peroxidase (CCP). Ferric APX at neutral pH is a mixture of 6- and 5-coordinate high-spin and 6-c low-spin hemes, the latter two species being dominant. The results suggest that the low-spin form derives from a water/hydroxo ligand bound to the heme iron and not from a strong internal ligand as observed in CCP at alkaline pH. Two Fe-Im stretching modes are identified, as in CCP, but the RR frequencies confirm a weaker His163-Asp208 hydrogen bond than in CCP, as suggested on the basis of the X-ray structure [Patterson, W. R., and Poulos, T. L. (1995) Biochemistry 34, 4331-4341]. The data show that CCP and APX have markedly different orientations of the vinyl substituents on the heme chromophore resulting from different steric constraints exerted by the protein matrix.