The reduction of glyceraldehyde by human erythrocytes. L-hexonate dehydrogenase activity.

Incubation of red cell suspensions with D-glyceraldehyde resulted in disappearance of glyceraldehyde and appearance of glycerol. Concomitantly, there was an increase of CO(2) formation from glucose. This indicated that the reduction of glyceraldehyde to glycerol occurred through a NADPH-linked system. Studies in hemolysates revealed the presence of an enzyme with the capacity to catalyze the reduction of glyceraldehyde to glycerol by NADPH. This enzyme was partially purified by DEAE chromatography. The elution pattern of the enzyme and its kinetic characteristics indicated that the enzyme was L-hexonate dehydrogenase (L-gulonate: NADP oxidoreductase, EC 1.1.1.19), not aldose reductase (Alditol: NADP oxidoreductase, EC 1.1.1.21), which had previously been thought present in erythrocytes. The reduction of glyceraldehyde to glycerol is one of a number of pathways for the metabolism of glyceraldehyde that have been found in red cells and/or other mammalian tissues.

Rational engineering of activity and specificity in a serine protease.

The discovery of the Na(+)-dependent allosteric regulation in serine proteases makes it possible to control catalytic activity and specificity in this class of enzymes in a way never considered before. We demonstrate that rational site-directed mutagenesis of residues controlling Na+ binding can profoundly after the properties of a serine protease. By suppressing Na+ binding to thrombin, we shift the balance between procoagulant and anticoagulant activities of the enzyme. Those mutants, compared to wild-type, have reduced specificity toward fibrinogen, but enhanced or slightly reduced specificity toward protein C. Because this engineering strategy targets a fundamental regulatory mechanism, it is amenable of extension to other enzymes of biological and pharmacological importance.

Critical role of W60d in thrombin allostery.

Mutation of residue W60d of thrombin, located 17 A from the Na+ binding site, suppresses Na+ binding and the functional differences between the slow and fast forms. The molecular basis for the long-range effect of this mutation is provided by a conspicuous network of water molecules which connects the Na+ binding environment to the specificity sites S1 and S2 of the enzyme. The mutation appears to stabilize thrombin in a hybrid conformation that is overall similar to the slow form, but with the fibrinogen recognition site functioning as in the fast form. It also affects the switch in specificity from fibrinogen to protein C linked to the release Na+ and the fast-->slow conversion. Under physiological conditions of pH, temperature and NaCl concentration, the W60dS mutant behaves as an anticoagulant. It has a reduced activity toward fibrinogen by 22-fold, while the reduction of protein C activation in the presence of saturating concentrations of thrombomodulin is less than 2-fold. Even more remarkable is the cleavage of fibrin I monomer leading to release of fibrinopeptide B, which is reduced by more than 130-fold. This property is reminiscent of the snake venom ancrod, which only releases fibrinopeptide A, and adds substantially to the anticoagulant potency of the W60dS mutant. In fact, the clotting time in the presence of this mutant is prolonged more than 40-fold compared to the wild-type.

A simple, rapid, efficient method for the preparation of gamma 32P-labeled guanosine triphosphate (GTP) and adenosine triphosphate (ATP).

A simplified new method for synthesis fo gamma 32P GTP and gamma 32P ATP is described. Radiophosphorus is incorporated into GDP and ADP to form GTP and ATP, respectively, in the phosphoglycerate kinase reaction. The product is separated by ion exchange resin chromatography. The procedure is simple, gives a product a high purity, and the label is confined to the gamma position.

Placental acid hydrolase purification on concanavalin A-sepharose.

Crude human placental extract was chromatographed on concanavalin A-Sepharose. Using a single procedure, 20- to over 300-fold purification of six acid glycohydrolases was achieved, in most cases with a good yield. Improved yield of individual enzymes could be achieved by modifications of the method of elution. The capacity of concanavalin A-Sepharose to bind glycohydrolases from placenta was very large, and will provice a useful procedure for the large-scale purification of the human enzymes.

Large heat capacity change in a protein-monovalent cation interaction.

Current views about protein-ligand interactions state that electrostatic forces drive the binding of charged species and that burial of hydrophobic and polar surfaces controls the heat capacity change associated with the reaction. For the interaction of a protein with a monovalent cation the electrostatic components are expected to be significant due to the ionic nature of the ligand, whereas the heat capacity change is expected to be small due to the size of the surface area involved in the recognition event. The physiologically important interaction of Na+ with thrombin was studied over the temperature range from 5 to 45 degrees C and the ionic strength range from 50 to 800 mM. These measurements reveal an unanticipated result that bears quite generally on studies of molecular recognition and protein folding. Binding of Na+ to thrombin is characterized by a modest dependence on ionic strength but a large and negative heat capacity change of -1.1 +/- 0.1 kcal mol-1 K-1. The small electrostatic coupling can be explained in terms of a minimal perturbation of the ionic atmosphere of the protein upon Na+ binding. The large heat capacity change, however, is difficult to reconcile with current views on the origin of this effect from surface area changes or large folding transitions coupled to binding. It is proposed that this change is linked to burial of a large cluster of water molecules in the Na+ binding pocket upon Na+ binding. Due to their reduced mobility and highly ordered structure, water molecules sequestered in the interior of a protein must have a lower heat capacity compared to those on the surface of a protein or in the bulk solvent. Hence, a binding or folding event where water molecules are buried may result in significant heat capacity changes independent of changes in exposed hydrophobic surface or coupled conformational transitions.

Formation of a calcium-binding site on bovine activated factor V following recombination of the isolated subunits.

Calcium binding to the activated form of blood coagulation Factor V (Factor Va) has been studied by equilibrium dialysis. A single calcium-binding site was observed with a dissociation constant of 24 +/- 4.0 microM. Unlike Factor V, Factor Va consists of two subunits. Following separation and reconstitution of the isolated subunits, a single calcium-binding site with a similar dissociation constant (24 +/- 2.0 microM) was again observed. No calcium binding was detected to either isolated subunit of Factor Va. Recombination of the Factor Va subunits used in the above equilibrium binding studies resulted in reformation of the calcium-binding site. Gel filtration experiments indicated that occupancy of this site with Ca2+ was required to maintain high affinity association between the Factor Va subunits. These data indicate that the calcium-binding site is either formed or stabilized by subunit-subunit interaction and that occupancy of this single site is sufficient to stabilize the intersubunit interactions.

Human red cells protein kinase in normal subjects and patients with hereditary spherocytosis, sickle cell disease, and autoimmune hemolytic anemia.

A somewhat simplified modification of a previously described method for the measurement of red cell membrane phosphorylation by ATP has been devised. Phosphorylation of membranes was linear with time for only 5-10 min, and linearity with membrane concentration was observed only when assays were limited to short incubation times. Protein kinase activity of hereditary spherocytosis (HS) membranes was found to be normal. However, the average phosphorylation after 60 min incubation was less in HS membranes than in normal membranes. Findings similar to those in HS membranes were observed in sickle cell disease. The Km of red cell protein kinase for ATP is approximately 10(-5) M. Membrane phosphate binding sites are not saturated in either HS or normal membranes after 1 hr incubation with ATP. Approximately 27% of phosphorylating activity is lost after 1 hr incubation at 37 degrees C. GTP is a very inefficient phosphate donor. Under the conditions of measurement employed, the enzyme is slightly stimulated by 1 muM cAMP, but is not stimulated by 1 muM cGMP. Dephosphorylation of red cell membranes after labeling occurs at a similar rate in HS as in normal membranes. Although a mild abnormally in membrane phosphorylation is observed in HS, this could not be demonstrated to be due to a decrease in protein kinase activity or in alterations of its kinetic properties. The abnormally seen is not specific for HS.

Loss of prothrombin and of factor Xa-factor Va interactions upon inactivation of factor Va by activated protein C.

Activated factor V (factor Va) is composed of two nonidentical subunits which can be dissociated on chelation of the bound Ca2+ with EDTA. The isolated subunits can be recombined in the presence of Ca2+ to form factor Va. The factor Va heavy chain (Mr = 94,000) binds to prothrombin in a specific and Ca2+-independent fashion. Following inactivation of either factor Va or the factor Va heavy chain by limited proteolysis with activated protein C, factor Va no longer binds to the immobilized prothrombin. Factor Va also binds specifically to (p-amidinophenyl)-methanesulfonyl-factor Xa-Affi-Gel 15. However, neither isolated subunit binds to this column. Factor Va inactivated by activated protein C is no longer retained by the factor Xa column. This data suggests that both subunits are required for optimal factor Va-factor Xa interaction and that inactivation of factor Va with activated protein C reduces the affinity of factor Va for both prothrombin and factor Xa.

Existence of only a single functional pool of adenosine triphosphate in human erythrocytes.

The question of whether separate "membrane" and "soluble" pools of ATP exist in erythrocytes has been examined. Phosphoglycerate kinase (EC 2.7.2.3)-derived ("membrane") ATP was labeled by short-term incubation with inorganic [32P]phosphate. Pyruvate kinase (EC 2.7.1.40)-derived ("soluble")ATP is not labeled under these circumstances. The specific activity of the gamma-phosphate of "soluble" ATP was then evaluated by the addition of 2-deoxyglucose and measurement of the specific activity of 2-deoxyglucose-6-[32P]phosphate formed. This specific activity was essentially the same as the overall specific activity of erythrocyte ATP gama-phosphate, indicating that no functional pools of phosphoglycerate kinase-derived and pyruvate kinase-derived ATP exist in erythrocytes.

Identification of residues linked to the slow-->fast transition of thrombin.

Residues energetically linked to the allosteric transition of thrombin from its anticoagulant slow form to the procoagulant fast form have been identified by site-directed mutagenesis. The energetics of recognition by the two forms of the enzyme were probed by using a synthetic chromogenic substrate, fibrinogen, and hirudin. The thrombin residues E39, W60d, E192, D221, and D222 are linked to the slow-->fast transition and are part of an "allosteric core" through which events originating at the Na+ binding loop propagate to other regions of the enzyme. The thrombin residues Y76, W96, W148, and R173 lie at the periphery of the allosteric core, affect recognition of fibrinogen and hirudin to the same extent in both forms, and are not linked to the slow-->fast transition.

Unexpected crucial role of residue 225 in serine proteases.

Residue 225 in serine proteases of the chymotrypsin family is Pro or Tyr in more than 95% of nearly 300 available sequences. Proteases with Y225 (like some blood coagulation and complement factors) are almost exclusively found in vertebrates, whereas proteases with P225 (like degradative enzymes) are present from bacteria to human. Saturation mutagenesis of Y225 in thrombin shows that residue 225 affects ligand recognition up to 60,000-fold. With the exception of Tyr and Phe, all residues are associated with comparable or greatly reduced catalytic activity relative to Pro. The crystal structures of three mutants that differ widely in catalytic activity (Y225F, Y225P, and Y225I) show that although residue 225 makes no contact with substrate, it drastically influences the shape of the water channel around the primary specificity site. The activity profiles obtained for thrombin also suggest that the conversion of Pro to Tyr or Phe documented in the vertebrates occurred through Ser and was driven by a significant gain (up to 50-fold) in catalytic activity. In fact, Ser and Phe are documented in 4% of serine proteases, which together with Pro and Tyr account for almost the entire distribution of residues at position 225. The unexpected crucial role of residue 225 in serine proteases explains the evolutionary selection of residues at this position and shows that the structural determinants of protease activity and specificity are more complex than currently believed. These findings have broad implications in the rational design of enzymes with enhanced catalytic properties.

Glu192-->Gln substitution in thrombin yields an enzyme that is effectively inhibited by bovine pancreatic trypsin inhibitor and tissue factor pathway inhibitor.

Modeling studies have ascribed the remarkable resistance of thrombin to inhibition by the Kunitz type inhibitors, bovine pancreatic trypsin inhibitor (BPTI), and tissue factor pathway inhibitor (TFPI), to steric inhibition by the 60-loop insertion, especially Trp60D (in the chymotrypsin numbering system). Indeed, deletion of Pro60B, Pro60C, and Trp60D from this loop (des-PPW) enhances BPTI inhibition (Ki = 16 nM) (Le Bonniec, B. F., Guinto, E. R., MacGillivray, R. T. A., Stone, S. R., and Esmon, C. T. (1993) J. Biol. Chem. 268, 19055-19061). Activated protein C, however, lacks an equivalent insertion loop but is nevertheless resistant to inhibition by these Kunitz inhibitors. A unique feature of thrombin and activated protein C is the presence of Glu at position 192. Substitution of Glu192 with Gln in activated protein C dramatically enhances inhibition by BPTI and TFPI (Rezaie, A. and Esmon, C. T. (1993) J. Biol. Chem. 268, 19943-19948). We now demonstrate that thrombin E192Q (the Glu192-->Gln mutant) is inhibited by BPTI (Ki = 24 nM) or TFPI (Ki = 14 nM) much more effectively than wild type thrombin (Ki > 1 microM for both inhibitors). A thrombin mutant having both the des-PPW deletion and E192Q substitution binds BPTI (Ki = 35 pM) and TFPI (Ki = 25 pM) even tighter. BPTI can displace dansylarginine N-(-3-ethyl-1,5-pentanediyl)-amide from the active site of thrombin E192Q (Ki = 19 nM), indicating that BPTI interacts directly with the S1 binding site in thrombin. The E192Q mutation and PPW deletion contribute comparably and additively to the binding energy of thrombin with the Kunitz inhibitors. We suggest that access to the active center of thrombin is less restricted than predicted from previous studies.

Identification of thrombin residues that modulate its interactions with antithrombin III and alpha 1-antitrypsin.

The role of thrombin's catalytic groove in the interaction with serpin has been investigated by comparing the association rate constant (kon) of several mutated thrombins with various serpins. The results indicated that Glu192, located three residues prior to the catalytic serine, and the major insertion in the sequence of thrombin compared with trypsin (residues Tyr60A-Trp60D) play an important role in modulating thrombin's interactions with serpins. Replacement of Glu192 by glutamine increased by 3 orders of magnitude the kon value with alpha 1-antitrypsin (which has a P1 methionine) but did not markedly alter the kon value with serpins containing a P1 arginine. The des-PPW thrombin mutant (lacking residues Pro60B, Pro60C, and Trp60D) exhibited a similar kon value as thrombin with protease nexin-1 but a kon value 2 orders of magnitude lower with antithrombin III. Thus, the 60-loop insertion of thrombin appears critical for its interaction with antithrombin III but dispensable for the formation of a complex with protease nexin-1. Heparin increased markedly the kon values for antithrombin III and protease nexin-1 with all thrombin variants tested, but a more dramatic effect was observed with a thrombin mutant (des-ETW) lacking residues Glu146, Thr147, and Trp148 (on the opposite side of the catalytic site relative to the 60-loop insertion). At the optimum concentration, heparin increased the kon value of the des-ETW--antithrombin III interaction by nearly 5 orders of magnitude, considerably more than for thrombin, suggesting that heparin is able to compensate in part for the adverse effects of the des-ETW mutation on the structure of thrombin.

Interaction of thrombin des-ETW with antithrombin III, the Kunitz inhibitors, thrombomodulin and protein C. Structural link between the autolysis loop and the Tyr-Pro-Pro-Trp insertion of thrombin.

X-ray diffraction studies of human thrombin revealed that compared with trypsin, two insertions (B and C) potentially limit access to the active site groove. When amino acids Glu146, Thr147, and Trp148, adjacent to the C-insertion (autolysis loop), are deleted the resulting thrombin (des-ETW) has dramatically altered interaction with serine protease inhibitors. Whereas des-ETW resists antithrombin III inactivation with a rate constant (Kon) approximately 350-fold slower than for thrombin, des-ETW is remarkably sensitive to the Kunitz inhibitors, with inhibition constants (Ki) decreased from 2.6 microM to 34 nM for the soybean trypsin inhibitor and from 52 microM to 1.8 microM for the bovine pancreatic trypsin inhibitor. The affinity for hirudin (Ki = 5.6 pM) is weakened at least 30-fold compared with recombinant thrombin. The mutation affects the charge stabilizing system and the primary binding pocket of thrombin as depicted by a decrease in Kon for diisopropylfluorophosphate (9.5-fold) and for N alpha-p-tosyl-L-lysine-chloromethyl ketone (51-fold) and a 39-fold increase in the Ki for benzamidine. With peptidyl p-nitroanilide substrates, the des-ETW deletion results in changes in the Michaelis (Km) and/or catalytic (kcat) constants, worsened as much as 85-fold (Km) or 100-fold (kcat). The specific clotting activity of des-ETW is less than 5% that of thrombin and the kcat/Km for protein C activation in the absence of cofactor less than 2%. Thrombomodulin binds to des-ETW with a dissociation constant of approximately 2.5 nM and partially restores its ability to activate protein C since, in the presence of the cofactor, kcat/Km rises to 6.5% that of thrombin. This study suggests that the ETW motif of thrombin prevents (directly or indirectly) its interaction with the two Kunitz inhibitors and is not essential for the thrombomodulin-mediated enhancement of protein C activation.

Nonenzymatic conversion of human hexosaminidase A.

Incubation of purified hexosaminidase A with merthiolate, parahydroxymercuribenzoic acid, or silver ions resulted in the formation of an enzyme which was identical, in all respects tested, with hexosaminidase B. Its electrophoretic mobility was identical to hexasaminidase B at three different pH levels. Its chromatographic properties, thermostability, and immunologic reactivity with specific anti-hexosaminidase A and anti-hexosaminidase B antisera were indistinguishable from hexosaminidase B. Conversion could be prevented by GSH but was not reversed by GSH once it had occurred. Conversion of hexosaminidase A to hexosaminindase B was accompanied by the appearance of an electrophoretically rapid, catalytically inactive protein. Hexosaminidase B, itself, was unaltered by incubation with merthiolate. These findings support a previously proposed model of hexosaminidase structure in which hexosaminidase A is (alphabeta)3 and hexosamindase B is (betabeta)2. On treatment of hexosaminidase A with merthiolate and other converting agents alpha and beta subunits are presumably dissociated and they reassociate as (betabeta)3, that is, hexosaminidase B. The expected free or polymerized catalytically inactive, alpha chains are detected on acrylamide gel electrophoresis. We suggest that the catalytic site of human hexosaminidase may be present only on the beta subunit and that the alpha subunit influences the substrate specificity of the enzyme, particularly as directed toward GM2.

Localization of the gene encoding human factor V to chromosome 1q21-25.

The gene encoding human coagulation Factor V (FV), one of the cofactors in the blood clotting process, has been mapped to chromosome 1 by both Southern hybridization to DNA from human-hamster somatic cell hybrids and in situ hybridization. The whole plasmid pUC3A containing a 1.5-kb cDNA sequence for FV was 32P-labeled for Southern analysis and 3H-labeled for in situ hybridization to metaphase chromosomes. The results localized the FV gene to the region of 1q21-25.

The complete cDNA sequence of bovine coagulation factor V.

Lack of availability of a primary structure for bovine factor V has hindered detailed analysis of a vast majority of structure-function correlations on this molecule. To determine the primary structure of bovine factor V, we used liver mRNA as a template for the synthesis of three cDNA libraries. The sequences of seven overlapping cDNA clones infer two bovine factor V variants. Variant 1 results in a 6910-basepair (bp) cDNA including 103 bp of 5'-untranslated sequence, 6633 bp of coding sequence and 171 bp of 3'-untranslated sequence with a putative polyadenylation site. Variant 2 differs only in the size of the coding sequence (6618 bp). The open reading frame translates to factor V consisting of 2211 (or 2206) amino acids including a 28-amino acid signal peptide. Comparison of the amino acid sequences with human factor Va reveals 84% identity for the heavy and 86% for the light chains. In contrast, the B domain (connecting region) exhibits only 59% identity relative to the human molecule. The bovine B domain contains two repeats of a 14-amino acid structure that is contained only once in the human sequence. Bovine factor V lacks one of the nine amino acid repeats and one of the 17 amino acid repeats present in the human B domain. Factor V has little homology to the factor VIII molecule in the B domain. The 17-amino acid repeat missing in bovine factor V allows identification of an 18-amino acid sequence that is homologous to the B domain of human factor VIII. These 18 amino acids may either constitute the unique vestige of a divergent evolution between the B domains of factors V and VIII or reveal the convergent evolution toward a critical epitope involved in the activation of both procofactors.