Role of non-covalent enzyme‒substrate interactions in the reaction catalysed by cellobiose phosphorylase from Cellulomonas uda

Abstract

Steady-state kinetic studies of the enzymic glucosyl transfer to and from phosphate catalysed by cellobiose phosphorylase from Cellulomonas uda have shown that this enzyme operates by a ternary-complex kinetic mechanism in which beta-cellobiose binds before phosphate, and beta-D-glucose and alpha-D-glucopyranosyl phosphate are released in that order. alpha-D-Glucopyranosyl fluoride (but not beta-D-glucopyranosyl fluoride) serves as alternative glucosyl donor for beta-cellobiose synthesis with a specificity constant that is one-ninth that of the corresponding enzymic reaction with alpha-D-glucopyranosyl phosphate (approximately 20000 M(-1).s(-1) at 30 degrees C). The kinetic parameters for a complete series of deoxy and deoxyfluoro analogues of D-glucose have been determined and the data yield estimates of the net strengths of hydrogen-bonding interactions with the non-reacting hydroxy groups of D-glucose at the transition state (0.8-4.0 kcal/mol, where 1 cal identical with 4.184 J) and enable the prediction of the polarities of these hydrogen bonds. Each hydroxy group functions as donor of a hydrogen for bonding to probably a charged (at 3-OH) or neutral (at 2-OH and 6-OH) acceptor group on the enzyme. The equatorial 1-OH is essential for enzyme activity. Derivatives of D-glucose in which the 1-OH or the reacting 4-OH were replaced by hydrogen or fluorine have been tested as inhibitors to measure their affinities for the sugar-binding subsite +1 (numbered from the bond-cleaving/forming site). The data show that hydrogen-bonding interactions between the 1-OH and 4-OH and charged groups on the enzyme stabilize the ground-state ternary complex of the enzymic synthesis of beta-cellobiose by 2.3 and 0.4 kcal/mol, respectively, and assist the precise positioning of beta-D-glucose for catalysis.

Transcriptional Analysis of Biofilm Formation Processes in the Anaerobic, Hyperthermophilic BacteriumThermotoga maritima

“…Induction of an NAD ϩ -dependent family 4 ␣-glucuronidase (TM0752, 3.1-fold) (99) and a ␤-galactosidase (TM0310, 2.9-fold) was observed in the (126), which it converts to D-glucose and glucose-1-phosphate (68). Characterization of the T. maritima homolog revealed substrate specificity for cellobiose in the hydrolysis reaction but relaxed synthetic specificity for the reverse reaction, allowing mannose, xylose, glucosamine, 2-and 6-deoxy-D-glucose, and ␤-D-glucoside to act as glucosyl acceptors for glucose-1-P (79).…”

Identification of Lacto-N-Biose I Phosphorylase fromVibrio vulnificusCMCP6

“…2). These results indicate that the phosphorolytic reaction of GalGlyNAcP Vv followed the sequential bi-bi mechanism, like the reactions of inverting phosphorylases, such as GalGlyNAcP from B. bifidum (5), GalGlyNAcP Bl (27), GalGlyNAcP Cp (27), maltose phosphorylase (39), cellobiose phosphorylase (15,17,28,34), chitobiose phosphorylase (12), and laminaribiose phosphorylase (18).…”

Development and application of a screening assay for glycoside phosphorylases

“…Although several phosphatase inhibitors have been described in literature, most of them are not useful here because they can also inhibit phosphorylase enzymes, or because they are incompatible with the phosphate determination method. For example, vanadate was shown to be an inhibitor of phosphatases [15] but also inhibits CP activity [13]. Fluoride and molybdate, both shown to be potent phosphatase inhibitors [15; 16; 17], were tested at different concentrations for inhibition of phosphatase activity in E. coli/pXCP cell extracts.…”