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Biomedical subjects

S Kirkwood

Publications and source records attributed to S Kirkwood.

At least 19 recordsLinked to original sources
PubMed1987

Implication of histidine at the active site of exo-beta-(1-3)-D-glucanase from Basidiomycete sp. QM 806.

The enzyme, exo-beta-(1-3)-D-glucanase, (EC 3.2.1-) obtained from a culture filtrate of Basidiomycete sp. QM 806, has been obtained in a highly purified form and preliminary investigations on its mechanism of action have been reported (Peterson, D. R., and Kirkwood, S. (1975) Carbohydr. Res. 41, 273-283). Studies reported in this paper, have provided strong evidence for the role of histidine in the catalytic site of this carbohydrase. Chemical modifications of the amino acid residues in the enzyme with diazotized 5-amino-1H-tetrazole or tetranitromethane caused irreversible loss of enzyme activity which varied according to the time of exposure to, or concentration of the inhibitor. Prior incubation of the enzyme with a substrate considerably reduced the extent of this inhibition. Amino acid analysis of the enzyme treated in these ways clearly indicated that the substrate protected histidine residues from chemical modification by the diazotized 5-amino-1H-tetrazole. Chemical modification of both histidine and tyrosine residues were effected by incubating the enzyme with the inhibitors described above. Although evidence is presented to suggest that tyrosine is not directly involved in the active site of the enzyme (the catalytic site or the binding site), the role of this residue in the maintenance of the enzyme conformation is discussed. Enzyme assays carried out either in aqueous or deuterated buffer systems provided further evidence which is consistent with the proposed enzyme mechanism.

Amino Acids↗
PubMed1984

Cellulose digestion by domestic turkeys fed low or high fiber diets.

To determine whether dietary preconditioning might improve fibrolysis in turkeys, Wrolstad Medium white hens were fed either a high (16.13%, HF) or low (2.80%, LF) fiber diet for 6-60 weeks. The substrate to test for cellulolysis, [14C]cellulose, was introduced into the gizzard per os or into one cecum via a cannulated fistula. Immediately after [14C]cellulose introduction, turkeys were placed in an airtight chamber (0.3 m3) from which exhaled CO2 was collected and analyzed for 14CO2. An average of 10.4% of the 14C was recovered in CO2 from turkeys fed the HF diet, and only 2.9% was recovered from the LF-fed birds that were orally dosed. In turkeys dosed via cecal cannula, recoveries averaged 15.4 and 2.9% for HF- and LF- fed birds, respectively. Recoveries were 7.0 and 1.4% in HF- and LF-fed turkeys that were cecectomized. Preconditioning to an HF diet does appear to improve cellulolysis.

Animals↗
PubMed1979

Mechanisms of action of histidinol dehydrogenase and UDP-Glc dehydrogenase. Evidence that the half-reactions proceed on separate subunits.

Histidinol dehydrogenase and UDP-Glc dehydrogenase catalyze 4-electron dehydrogenations that convert primary alcohol groups to the corresponding acids. Both reactions proceed in two distinct steps involving the oxidation of the primary alcohol to a bound form of the intermediate aldehyde, followed by oxidation of this to the corresponding acid. The enzymes have subunit structure, the former is made up of two subunits and the latter of six (beef liver enzyme). Evidence is presented that the two half-reactions proceed independently of the overall reaction. Histidinol dehydrogenase preparations that approach total dissociation into subunits also approach total inhibition of the overall reaction, while the second half reaction is completely unaffected and 50% of the first half-reaction survives. Further, the fraction of overall activity surviving in partially dissociated preparations follows the weight fraction of residual dimer. UDP-Glc dehydrogenase behaves in an analogous fashion. These data are interpreted on the basis that both enzymes function by carrying out first oxidation step at a site on one subunit and then pass the intermediate to a vicinal site on the adjacent subunit, where the reaction is completed.

Alcohol Oxidoreductases↗
PubMed1978

Purification, characterization, and action-pattern studies on the endo-(1 linked to 3)-beta-D-glucanase from Rhizopus arrhizus QM 1032.

The extracellular (1 linked to 3)-beta-D-glucanase [(1 linked to 3)-beta-D-glucan glucanohydrolase, EC 3.2.1.6] produced by Rhizopus arrhizus QU 1032 was purified 305-fold in 70% overall yield. This preparation was found to be homogeneous by ultracentrifugation (sedimentation velocity and equilibrium studies), electrophoresis on acrylamide gel with normal, sodium dodecyl sulfate, and urea-acetic acid gels, and upon isoelectric focusing. The amino acid composition of the enzyme has been determined and it possesses a carbohydrate moiety compose of mannose and galactose (in the ratio approximately 5:1) that is linked to the protein through a 2-acetamido-2-deoxyglucose residue. The molecular weight, as determined by equilibrium sedimentation, is 28,800 and this number was confirmed by electrophoresis on gels of sodium dodecyl sulfate. The enzyme does not possess subunit structure. It hydrolyzes its substrates with retention of configuration and possesses transglycosylating ability. The rates of hydrolysis of a wide variety of substrates were determined, and its action pattern on a series of oligosaccharides containing mixed (1 linked to 3)-, (1 linked to 4)-, and (1 linked to 6)-beta-D-glucopyranosyl residues was investigated. The enzyme favors stretches of beta-D-(1 linked to 3) linkages, but it can hydrolyze beta-D-(1 linked to 4) linkages that are flanked on the non-reducing side with stretches of beta-D-(1 linked to 3) links. The enzyme will not act on (1 linked to 6)-beta-D-glucosyl linkages located in stretches of beta-D-(1 linked to 3) and will not act on (1 linked to 3) beta-D-glycosidic linkages involving sugars other than D-glucose.

Glucan Endo-1,3-beta-D-Glucosidase↗
PubMed1977

UDPglucose dehydrogenase. Kinetics and their mechanistic implications.

Initial velocity and product inhibition studies were carried out on UDP-glucose dehydrogenase (UDPglucose: NAD+ 6-oxidoreductase, EC 1.1.1.22) from beef liver to determine if the kinetics of the reaction are compatible with the established mechanism. An intersecting initial velocity pattern was observed with NAD+ as the variable substrate and UDPG as the changing fixed substrate. UDPglucuronic acid gave competitive inhibition of UDPG and non-competitive inhibition of NAD+. Inhibition by NADH gave complex patterns.Lineweaver-Burk plots of 1/upsilon versus 1/NAD+ at varied levels of NADH gave highly non-linear curves. At levels of NAD+ below 0.05 mM, non-competitive inhibition patterns were observed giving parabolic curves. Extrapolation to saturation with NAD+ showed NADH gave linear uncompetitive inhibition of UDPG if NAD+ was saturating. However, at levels of NAD+ above 0.10 mM, NADH became a competitive inhibitor of NAD+ (parabolic curves) and when NAD+ was saturating NADH gave no inhibition of UDPG. NADH was non-competitive versus UDPG when NAD+ was not saturating. These results are compatible with a mechanism in which UDPG binds first, followed by NAD+, which is reduced and released. A second mol of NAD+ is then bound, reduced, and released. The irreversible step in the reaction must occur after the release of the second mol of NADH but before the release of UDPglucuronic acid. This is apparently caused by the hydrolysis of a thiol ester between UDPglucoronic acid and the essential thiol group of the enzyme. Examination of rate equations indicated that this hydrolysis is the rate-limiting step in the overall reaction. The discontinuity in the velocities observed at high NAD+ concentrations is apparently caused by the binding of NAD+ in the active site after the release of the second mol of NADH, eliminating the NADH inhibition when NAD+ becomes saturating.

Alcohol Oxidoreductases↗
PubMed1977

Mechanism of action of uridine diphoglucose dehydrogenase. Evidence for an essential lysine residue at the active site.

The oxidation of UDP-glucose by the enzyme UDP-glucose dehydrogenase (EC 1.1.1.22) from beef liver has been shown to proceed via the enzyme-bound intermediate, UDP-alpha-D-glyco-hexodialdose. The enzyme does not release this aldehyde, nor can it be trapped by reaction with hydroxylamine, thiosemicarbazide, or cyanide. Tight binding of the intermediate aldehyde can be explained by the recent observation that the essential thiol group of the enzyme forms a thiohemiacetal with the aldehyde during the course of the reaction. However, an enzyme preparation with the essential thiol derivatized with cyanide will still not release the aldehyde, indicating an additional as yet unknown binding mechanism. Derivatization ([14C]formaldehyde, followed by NaBH4 reduction) of 6 of the approximately 168 lysine residues per enzyme molecule (of six catalytic subunits) results in destruction of 47% of the enzyme activity, suggesting the involvement of an essential reactive lysine in the mechanism. Preincubation of the enzyme with UDP-glucose decreases both the loss of activity and incorporation of the label, indicating that this lysine is in the vicinity of the active site. Acid hydrolysis of the labeled preparation, followed by paper chromatography, shows that the label has a mobility, in the system used, that is identical with lysine. Elution of this spot followed by chromatography on Aminex A-5 resin showed that it contained the expected mixture of epsilon-N-methyl lysines. When enzyme that has its essential thiol derivatized with cyanide is incubated with UDP-[14C]glucose and NAD+, and then reduced with NaB3H4, a stable enzyme complex is formed which contains both labels. Acid hydrolysis of this preparation, followed by either two-dimensional paper chromatography or separation in an amino acid analyzer, results in both labels appearing in the position of lysine. It is evident that the enzyme oxidizes the UDP-[14C]glucose to the corresponding aldehyde which occurs as the Schiff's base with an essential lysine. This is then reduced by the NaB3H4 to form a secondary amine which is stable toward hydrolysis and migrates with lysine in separation procedures. As would be predicted, the enzyme can be similarly labeled by treatment with UDP-alpha-D-gluco-hexodisidose alone, followed by NaB3H4 reduction. The same hydrolysis product results from this procedure, and it behaves identically with the product formed by treating alpha-N-acetyl lysine with UDP-alpha-D-gluco-hexodialdose, reducing with NaBH4, and then hydrolyzing. This substance appears to be N5-((5-formyl-2-furanyl)methyl)lysine. When chromatographed on Aminex A-5, both the model compound and enzyme hydrolysate gave peaks corresponding to free lysine and the proposed derivative. Evidence is presented that the oxidation of UDP-glucose to the aldehyde is a concerted reaction involving the formation of the Schiff's base, rather than the formation of the aldehyde with the subsequent formation of the Schiff's base...

Alcohol Oxidoreductases↗
PubMed1975

Mechanism of action of uridine diphosphoglucose dehydrogenase. Evidence for a second reversible dehydrogenation step involving an essential thiol group.

Although the enzyme UDP-glucose dehydrogenase from beef liver (E.C. 1.1.1.22) is known to abstract the pro-R hydrogen stereospecifically at carbon 6 of the glucose moiety of the substrate by a reversible step in converting UDP-glucose to UDP-alpha-D-gluco-hexodialdose (UDP-Glc-6-CHO), prolonged incubation of the enzyme with UDP-glucose and tritium-labeled NADH, under conditions favoring hydrogen exchange between the two, results in equivalent labeling of both hydrogens at carbon 6. This shows that the pro-S hydrogen at carbon 6 is also abstracted by a reversible process which must then involve a derivative of the carboxyl group of UDP-glucuronic acid (UDP-GlcUA) that is capable of reversible hydrogenation-dehydrogenation. It is the hydrolysis of this derivative that accounts for the well known irreversibility of the overall reaction. Derivatization of the enzyme's essential thiol group with 5,5'-dithiobis-(2-nitrobenzoate) eliminates the ability of the enzyme to either oxidize or reduce UDP-Glc-6-CHO. Replacement of the 5-thio-2-nitrobenzoate group with cyanide fully restores the enzyme's capacity to reduce UDP-Glc-6-CHO but has no effect on the inhibition of the oxidation to UDP-GlcUA. This indicates that the essential thiol group is involved in the second reversible dehydrogenation step and serves to form a thiol ester with the carboxyl of the product, UDP-GlcUA. It is suggested that thiol ester intermediates are a general characteristic of all 4-electron NAD-linked dehydrogenase reactions.

Alcohol Oxidoreductases↗
PubMed1975

Studies on the structure and mechanism of an exo-(1 yields 3)-beta-D-glucanase from Basidiomycete QM806.

A method for the large-scale production of a (1 yields 3)-beta-D-glucan glucohydrolase (EC 3.2.1.58) from the culture filtrate of Basidiomycete QM806 is described. The final preparation is homogeneous by disc electrophoresis under non-dissociating and denaturing conditions, by ultracentrifugation, and by isoelectric focusing. Various physical and chemical characteristics of the enzyme have been determined, including terminal amino acid residues, extinction coefficient, and stability to pH extremes. The N-terminal amino acids are leucine and serine (Sanger's method) and the C-terminal amino acids are alanine, serine, and glycine (hydrazinolysis). pH profile studies show that no group titrating in the region 2.5-8 is directly involved with substrate binding and that a single group having a pKa of 6.5 is involved in the catalysis. Photooxidation of the enzyme caused rapid inactivation. The pH-dependence of this photooxidation, and amino acid analysis of the photooxidized enzyme, indicate that decomposition of histidine is probably responsible for the loss of activity. Other chemical modifications performed were: treatment with hydrogen peroxide under acidic conditions, esterification with diphenyldiazomethane, and oxidation with N-bromosuccinimide. Oxidation with N-bromosuccinimide indicated that a tryptophan side-chain is involved in, but not necessary for, the catalytic activity.

Amino Acid Sequence↗