The nuclear lamins form a karyoskeleton providing structural rigidity to the nucleus. One member of the lamin family, lamin A, is first synthesized as a 74 kDa precursor, prelamin A. After the endopeptidase and methylation reactions which occur after farnesylation of the CAAX-box cysteine, there is a second endoproteolysis that occurs 15 amino acids upstream from the C-terminal farnesylated cysteine residue. Studies with knockout mice have implicated the enzyme Zmpste24 (Face-1) as a suitable candidate to perform one or both of these proteolytic reactions. Evidence has been presented elsewhere establishing that Zmpste24 possesses a zinc-dependent CAAX endopeptidase activity. In the present study, we confirm this CAAX endopeptidase activity with recombinant, membrane-reconstituted Zmpste24 and show that it can accept a prelamin A farnesylated tetrapeptide as substrate. To monitor the second upstream endoproteolytic cleavage of prelamin A, we expressed a 33 kDa prelamin A C-terminal tail in insect cells. We demonstrate that this purified substrate possesses a C-terminal farnesylated and carboxyl-methylated cysteine and, therefore, constitutes a valid substrate for assaying the second endoproteolytic step in lamin A maturation. With this substrate, we demonstrate that insect cell membranes bearing recombinant Zmpste24 can also catalyse the second upstream endoproteolytic cleavage.

Candida albicans glucosamine-6-phosphate (GlcN-6-P) synthase was purified to apparent homogeneity with 52% yield from recombinant yeast YRSC-65 cells efficiently overexpressing the GFA1 gene. The pure enzyme exhibited K m(Gln) ‫؍‬ 1.56 mM and K m(Fru-6-P) ‫؍‬ 1.41 mM and catalyzed GlcN-6-P formation with k cat ‫؍‬ 1150 min ؊1 . The isoelectric point of 4.6 ؎ 0.05 was estimated from isoelectric chromatofocusing. Gel filtration, native polyacrylamide gel electrophoresis, subunit cross-linking, and SDS-polyacrylamide gel electrophoresis showed that the native enzyme was a homotetramer of 79.5-kDa subunits, with an apparent molecular mass of 330 -340 kDa. Results of chemical modification of the enzyme by group-specific reagents established an essential role of a cysteinyl residue at the glutamine-binding site and histidyl, lysyl, arginyl, and tyrosyl moieties at the Fru-6-Pbinding site. GlcN-6-P synthase in crude extract was effectively inhibited by UDP-GlcNAc (IC 50 ‫؍‬ 0.67 mM). Purification of the enzyme markedly decreased the sensitivity to the inhibitor, but this could be restored by addition of another effector, glucose 6-phosphate. Binding of UDP-GlcNAc to the pure enzyme in the presence of Glc-6-P showed strong negative cooperativity, with n H ‫؍‬ 0.54, whereas in the absence of this sugar phosphate no cooperative effect was observed. Pure enzyme was a substrate for cAMP-dependent protein kinase, the action of which led to the substantial increase of GlcN-6-P synthase activity, correlated with an extent of protein phosphorylation. The maximal level of activity was observed for the enzyme molecules containing 1.21 ؎ 0.08 mol of phosphate/mol of GlcN-6-P synthase. Monitoring of GlcN-6-P synthase activity and its sensitivity to UDP-GlcNAc during yeast 3 mycelia transformation of C. albicans cells, under in situ conditions, revealed a marked increase of the former and a substantial fall of the latter.

A beta-glycosidase (Mr 50,000) from Spodoptera frxgiperda larval midguts was purified, cloned and sequenced. It is active on aryl and alkyl beta-glucosides and cellodextrins that are all hydrolyzed at the same active site, as inferred from experiments of competition between substrates. Sequence alignment indicates that the amino acids directly involved in the catalysis are El87 (proton donor -pKa 7.5) and E399 (nucleophile -pKa 4.9), whereas chemical modification using tetranitromethane and phenylglyoxal and tertiary structure data suggest that R97 and Y331 affect their ionization. kcat and Km values obtained for different p-nitrophe-no1 beta-glycosides and sequence alignment indicate that the glycone moiety is stabilized in the transition state by a hydrophobic region around the hydroxymethyl group and by hydrogen bonds between the other equatorial hydroxyls and polar amino acids (439, E451, H142, N186 and E399) from the -1 subsite. Ki values for a range of alkyl beta-glucosides and oligocellodextrins reveal that the aglycone binding site is a hydrophobic cleft made up of 3 subsites with a polar amino acid only at its first (+I) subsite.This work concerns the comparison of the kinetic constants (Vmax and Km) obtained by different methods applied to a twosubstrate two-product enzymatic reaction of two aspartate aminotransferase (AAT) isoenzymes from Lupinus albus L. AAT catalyses the reversible transfer of an amino group from aspartate to 2-oxoglutarate to form oxaloacetate and glutamate, following a non-sequential kinetic mechanism named ping-pong bi-bi. With this mechanism, the high number of required analytical determinations makes the repetition of measurements often impractical. The following methods were used to calculate the kinetic constants: direct linear plot, least-squares fit to a hyperbola (followed by a secondary plot), fitting of an adequate overall rate equation (Alberty equation) and the Lineweaver-Burk plot (for comparison purposes). Our results show that there are some differences in the calculated values using these different methods. The results obtained by fitting the Alberty equation to our data are very similar to the ones obtained by the Lineweaver-Burk method (that should not be used to determine kinetic constants). This is probably due to the difficulty in judging the appropriate weighting scheme to use in the fitting of the rate equation. The direct linear plot (with primary and secondary plots) is thus considered the best method, as it does not need a weighting scheme or replicate measurements. If the primary plot is obtained by the least-squares fit to a hyperbola, the kinetic constants deviate a little from the values calculated from the direct linear plot.