Distribution of lactic dehydrogenase in X-irradiated tissues.

Exposure to whole-body X-irradiation in rats causes a marked increase in total lactic dehydrogenase (LDH) activity and decrease in H-LDH/M-LDH ratio in serum and tissues, the maximum effect being observed on the 8th post-irradiation day. While there is an elevation of M-LDH isoenzyme, H-LDH remains relatively constant. Studies on incorporation of DL-leucine-l-14C into heart LDH isoenzymes also revealed increased biosynthesis of M-LDH isoenzyme. This indicates that an adaptive mechanism is operative in the X-irradiated rats, whereby, in order to augment anaerobic glycolysis, synthesis of M-LDH is stimulated. Administration of the radioprotector cystamine does not alter the effects of X-irradiation on serum LDH activity.     

Alterations in rat lactate dehydrogenase: age-related comparison of M- and H-LDH

Lactate dehydrogenase (LDH: EC 1.1.1.27) of skeletal muscle and heart was purified from young and old female albino rats. Properties of both types, muscle (M-LDH) and heart (H-LDH), were compared by using enzymes purified from skeletal muscle and hearts of young (22-weeks) and old (116-weeks) rats. M-LDH showed differences with respect to properties such as thermal stability and effects of pH, urea, and sodium sulfite. However, H-LDH did not show any significant alterations in enzyme properties.     

M-LDH serves as a sarcolemmal K(ATP) channel subunit essential for cell protection against ischemia

ATP-sensitive K(+) (K(ATP)) channels in the heart are normally closed by high intracellular ATP, but are activated during ischemia to promote cellular survival. These channels are heteromultimers composed of Kir6.2 subunit, an inwardly rectifying K(+) channel core, and SUR2A, a regulatory subunit implicated in ligand-dependent regulation of channel gating. Here, we have shown that the muscle form (M-LDH), but not heart form (H-LDH), of lactate dehydrogenase is directly physically associated with the sarcolemmal K(ATP) channel by interacting with the Kir6.2 subunit via its N-terminus and with the SUR2A subunit via its C-terminus. The species of LDH bound to the channel regulated the channel activity despite millimolar concentration of intracellular ATP. The presence of M-LDH in the channel protein complex was required for opening of K(ATP) channels during ischemia and ischemia-resistant cellular phenotype. We conclude that M-LDH is an integral part of the sarcolemmal K(ATP) channel protein complex in vivo, where, by virtue of its catalytic activity, it couples the metabolic status of the cell with the K(ATP) channels activity that is essential for cell protection against ischemia.     

Photolabelling of mutant forms of the S1 subunit of pertussis toxin with NAD+.

The S1 subunit of pertussis toxin catalyses the hydrolysis of NAD+ (NAD+ glycohydrolysis) and the NAD(+)-dependent ADP-ribosylation of guanine-nucleotide-binding proteins. Recently, the S1 subunit of pertussis toxin was shown to be photolabelled by using radiolabelled NAD+ and u.v.; the primary labelled residue was Glu-129, thereby implicating this residue in the binding of NAD+. Studies from various laboratories have shown that the N-terminal portion of the S1 subunit, which shows sequence similarity to cholera toxin and Escherichia coli heat-labile toxin, is important to the maintenance of both glycohydrolase and transferase activity. In the present study the photolabelling technique was applied to the analysis of a series of recombinant-derived S1 molecules that possessed deletions or substitutions near the N-terminus of the S1 molecule. The results revealed a positive correlation between the extent of photolabelling with NAD+ and the magnitude of specific NAD+ glycohydrolase activity exhibited by the mutants. Enzyme kinetic analyses of the N-terminal mutants also identified a mutant with substantially reduced activity, a depressed photolabelling efficiency and a markedly increased Km for NAD+. The results support a direct role for the N-terminal region of the S1 subunit in the binding of NAD+, thereby providing a rationale for the effect of mutations in this region on enzymic activity.     

Histochemical localization of lactate dehydrogenase isoenzymes in human skeletal muscle fibers.

Lactate dehydrogenase (LDH) activity was histochemically localized in fibers of the vastus lateralis muscle of men and for comparative purpose in the soleus and plantaris muscleo of rats. Human muscle fibers were identified as fast twitch (FT) or slow twitch (ST) from the histochemical stain for myofibrillar adenosine triphosphatase activity. Rat skeletal muscle fibers were classified as fast-twitch-oxidative-glycolytic (FOG), fast-twitch-glycolytic (FG), or slow-twitch-oxidative (SO) on the basis of NADH-diaphorase and myofibrillar adenosine triphosphatase activities. Heart-type (H) LDH was identified by inhibition of the muscle-type (M) isozyme with 4 M urea. Total LDH as estimated histochemically was highest in the human FT and rat FG fibers. This was predominantly the M-LDH isozyme. ST fibers of human and SO fibers of rat skeletal muscle had the least total LDH but the most H-LDH activity. The FOG fibers of rat muscle contained a total LDH activity intermediate to that of the FG and SO fibers and a combination of H- and M-LDH. There were no fibers in the human muscle samples studied that had LDH activities similar to the FOG fibers of rat muscle.     

M-LDH serves as a regulatory subunit of the cytosolic substrate-channelling complex in vivo

Nucleoside diphosphate kinase A (NDPK-A) regulates the alpha1 isoform of the AMP-activated protein kinase (AMPK alpha1) selectively, independent of [AMP] and surrounding [ATP], by a process termed substrate channelling. Here, we show, using a range of empirically validated biochemical techniques, that the muscle form (M-LDH or LDH-A) and the heart form (H-LDH or LDH-B) of lactate dehydrogenase are physically associated with the liver cytosolic substrate-channelling complex such that M-LDH associates with NDPK-A, AMPK alpha1 and casein kinase 2 (CK2), whereas H-LDH associates with local NDPK-B. We find that the species of LDH bound to the substrate-channelling complex regulates the in vivo enzymatic activities of both AMPK and CK2, and has a downstream effect on the phospho-status of acetyl CoA carboxylase, a key regulator of cellular fat metabolism known to be a part of the cytosolic substrate-channelling complex in vivo. We hypothesise that the regulatory presence of LDH in the complex couples the substrate-channelling mechanism to both the glycolytic and redox states of the cell, allowing for efficient sensing of cell metabolic status, interfacing with the substrate-channelling complex and regulating the enzymatic activities of AMPK and CK2, two critical protein kinases.     

Lactate dehydrogenase isoenzymes in the eye, cardiac and skeletal muscles of several decapods]

Properties of lactate dehydrogenase (LDH) in the eye, heart and muscles of Hemigrapsus sanguineus, Paralithodes camtschatica, Erimacrus isenbeckii, Pandalus latirastrus, Pagurus brachiomastus have been studied with acrylamide gel electrophoresis and kinetics analysis. LDH in all the tissues of all the representatives studied was found to be specific for L-pyruvate and lactate; it migrated in electrophoresis as a single band revealing low mobility towards anode. The isoenzyme from P. camtschatica and P. latirastrus differed from the isoenzymes of other animals studied by higher mobility towards anode that reflected higher negative value of its total charge. The LDH isoenzymes in all the animals studied resembled the A4 (LDH5) of the vertebrates being unstable to the denaturing action of high temperature and being unaffected by high concentrations of pyruvate up to 1.0.10-3M. On the other hand, in conrast to the A4 of mammals, the LDH in question displayed enhancement of the reaction rate and decrease of the Km values upon increase in the NAD+ and NAD.H concentrations both in the presence of high or low lactate and pyruvate concentrations. The isoenzymes displayed catalytic activity also in the presence of NADP, the Km values for pyruvate in the presence of equimolar (2.25 mM) concentrations of NAD.H or NADP.H were practically identical and were found to be within the limits of 14-26.10-5 M. Molecular weight of the LDH studied assessed by the gel filtration method was found to be 130-140,000. It is suggested that the LDH isoenzyme from the representatives of the decapod crayfish studied is homologous in its certain properties to the homotetrameric A4 form of the vertebrates.     

Tyrosine modification of glucose dehydrogenase from Bacillus megaterium. Effect of tetranitromethane on the enzyme in the tetrameric and monomeric state

The active tetrameric glucose dehydrogenase from Bacillus megaterium is rapidly inactivated upon reaction with tetranitromethane. The inactivation is correlated with the nitration of a single tyrosine residue/subunit. The nitration does not influence the dissociation-reassociation process of the enzyme. The inactivation is prevented by the presence of NAD, AMP, ATP. The sequence around the nitrated tyrosine residue was determined and the residue was identified as Tyr-254 in the covalent structure of the enzyme. After dissociation of the enzyme into its monomers two tyrosine residues become susceptible to nitration. The nitrated subunits are unable to reassociate to the tetramer. Isolation and sequence analysis of the peptides containing nitrotyrosine indicated that two different tyrosine residues are predominantly modified. One residue is Tyr-254 which is essential for the catalytic activity and the other one is Tyr-160 which seems to be located in the subunit binding area.     

Identification of peptides in the adenine ring binding domain of glutamate and lactate dehydrogenase using 2-azido-NAD+.

Previously, the synthesis and validation of [32P]2N3NAD+ as an active site directed photoaffinity probe for glutamate dehydrogenase (GDH) was reported (8). This report shows that 2N3NAD+ is also an effective probe for the NAD+ binding site of lactate dehydrogenase (LDH). With the appropriate photolabeling procedures and immobilized boronate column chromatography the active site peptides of GDH and LDH involved in the adenine base binding domain have been isolated and sequenced. With both GDH and LDH a single photolabeled peptide, which contained the majority of the photoinserted radiolabel, was isolated. Additionally, these peptides had UV spectra that were markedly different from the nonphotolabeled peptides. The modified peptide from GDH corresponded to Cys270 through Lys289. Both sequencing and compositional analysis identified Glu275 as the site of photoinsertion. Sequencing of this peptide aborted at Glu275 after five rounds of analysis, indicating that insertion was blocking further progress. Compositional analysis showed that the entire sequence from residues 270 to 289 was present except that the single Glu residue was missing. This is interpreted as indicating that the photoinsertion is into the polypeptide backbone at the Glu site. The peptide isolated from LDH corresponded to Asp82 through Arg90. Sequencing of this peptide could be completed throughout with only the round at Tyr83 giving no identifiable residue. Compositional analysis of this peptide was in agreement with the peptide from Asp82 to Arg90 with the exception that the single Tyr residue was missing. This indicates that the photoinsertion is into the tyrosine side chain. This data was found to be in agreement with X-ray crystallographic results identifying the NAD(+)-binding domains.     

Crystal structure of S-adenosylhomocysteine hydrolase from rat liver.

The crystal structure of rat liver S-adenosyl-L-homocysteine hydrolase (AdoHcyase, EC 3.3.1.1) which catalyzes the reversible hydrolysis of S-adenosylhomocysteine (AdoHcy) has been determined at 2.8 A resolution. AdoHcyase from rat liver is a tetrameric enzyme with 431 amino acid residues in each identical subunit. The subunit is composed of the catalytic domain, the NAD+-binding domain, and the small C-terminal domain. Both catalytic and NAD+-binding domains are folded into an ellipsoid with a typical alpha/beta twisted open sheet structure. The C-terminal section is far from the main body of the subunit and extends into the opposite subunit. An NAD+ molecule binds to the consensus NAD+-binding cleft of the NAD+-binding domain. The peptide folding pattern of the catalytic domain is quite similar to the patterns observed in many methyltransferases. Although the crystal structure does not contain AdoHcy or its analogue, there is a well-formed AdoHcy-binding crevice in the catalytic domain. Without introducing any major structural changes, an AdoHcy molecule can be placed in the catalytic domain. In the structure described here, the catalytic and NAD+-binding domains are quite far apart from each other. Thus, the enzyme appears to have an "open" conformation in the absence of substrate. It is likely that binding of AdoHcy induces a large conformational change so as to place the ribose moiety of AdoHcy in close proximity to the nicotinamide moiety of NAD+. A catalytic mechanism of AdoHcyase has been proposed on the basis of this crystal structure. Glu155 acts as a proton acceptor from the O3'-H when the proton of C3'-H is abstracted by NAD+. His54 or Asp130 acts as a general acid-base catalyst, while Cys194 modulates the oxidation state of the bound NAD+. The polypeptide folding pattern of the catalytic domain suggests that AdoHcy molecules can travel freely to and from AdoHcyase and methyltransferases to properly regulate methyltransferase activities. We believe that the crystal structure described here can provide insight into the molecular architecture of this important regulatory enzyme.     

Purification and properties of NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase from spinach leaves.

An NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC. 1.2.1.12) has been purified from spinach leaves as a homogeneous protein of 150,000 daltons. Kinetic constants of 2.5 . 10(-4) M and 4 . 10(-4) M have been calculated for NAD+ and glyceraldehyde-3-phosphate, respectively. The amino acid composition is characterized by a cysteine content higher than that found in analogous enzymes. On sodium dodecyl sulphate gel electrophoresis, the native enzyme dissociates into two subunits of 37,000 and 14,000 daltons. The two subunits have been isolated in equimolar amounts by gel filtration; end-group analysis shows that alanine is the N-terminal residue of the large subunit, while serine is found at the N-terminus of the small subunit. Comparison of amino acid analysies and peptide maps shows that the two subunits have a different amino acid sequence. These results indicate that the NAD+-dependent glyceraldehyde-3-phosphate, dehydrogenase, isolated from spinach leaves has an atypical oligomeric structure, the protomer being formed by two different subunits.     

Crystal structure of a truncated mutant of glucose-fructose oxidoreductase shows that an N-terminal arm controls tetramer formation

N-terminal or C-terminal arms that extend from folded protein domains can play a critical role in quaternary structure and other intermolecular associations and/or in controlling biological activity. We have tested the role of an extended N-terminal arm in the structure and function of a periplasmic enzyme glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis. We have determined the crystal structure of the NAD(+) complex of a truncated form of the enzyme, GFORDelta, in which the first 22 residues of the N-terminal arm of the mature protein have been deleted. The structure, refined at 2.7 A resolution (R(cryst)=24.1%, R(free)=28.4%), shows that the truncated form of the enzyme forms a dimer and implies that the N-terminal arm is essential for tetramer formation by wild-type GFOR. Truncation of the N-terminal arm also greatly increases the solvent exposure of the cofactor; since GFOR activity is dependent on retention of the cofactor during the catalytic cycle we conclude that the absence of GFOR activity in this mutant results from dissociation of the cofactor. The N-terminal arm thus determines the quaternary structure and the retention of the cofactor for GFOR activity and during translocation into the periplasm. The structure of GFORDelta also shows how an additional mutation, Ser64Asp, converts the strict NADP(+) specificity of wild-type GFOR to a dual NADP(+)/NAD(+) specificity.     

Structure of human cytosolic X-prolyl aminopeptidase: a double Mn(II)-dependent dimeric enzyme with a novel three-domain subunit

X-prolyl aminopeptidases catalyze the removal of a penultimate prolyl residue from the N termini of peptides. Mammalian X-prolyl aminopeptidases are shown to be responsible for the degradation of bradykinin, a blood pressure regulator peptide, and have been linked to myocardial infarction. The x-ray crystal structure of human cytosolic X-prolyl aminopeptidase (XPN-PEP1) was solved at a resolution of 1.6 angstroms. The structure reveals a dimer with a unique three-domain organization in each subunit, rather than the two domains common to all other known structures of X-prolyl aminopeptidase and prolidases. The C-terminal catalytic domain of XPNPEP1 coordinates two metal ions and shares a similar fold with other prolyl aminopeptidases. Metal content analysis and activity assays confirm that the enzyme is double Mn(II) dependent for its activity, which contrasts with the previous notion that each XPNPEP1 subunit contains only one Mn(II) ion. Activity assays on an E41A mutant demonstrate that the acidic residue, which was considered as a stabilizing factor in the protonation of catalytic residue His498, plays only a marginal role in catalysis. Further mutagenesis reveals the significance of the N-terminal domain and dimerization for the activity of XPNPEP1, and we provide putative structural explanations for their functional roles. Structural comparisons further suggest mechanisms for substrate selectivity in different X-prolyl peptidases.     

Role of calpains and kininogens in inflammation.

Neutrophil chemotactic activity was found in the autodigest of calcium dependent cysteine proteinase (calpain) I purified from human erythrocytes, an active peptide was isolated, and its structure was determined. It was an N-acetyl nonapeptide with the sequence: N-acetyl Ser-Glu-Glu-Ile-Ile-Thr-Pro-Val-Tyr. This peptide was identical with the N-terminal amino acid sequence of the large subunit of calpain I deduced from cDNA sequence, except that the peptide was lacking a methionine residue and was acetylated at the N-terminus. A number of N-acetyl peptides with N-terminal amino acid sequences of large and small subunits of calpains I and II were synthesized and their chemotactic activity was estimated. In addition to the N-acetyl nonapeptide from calpain I large subunit, several peptides of different lengths from the small subunit showed dose-dependent migrations of neutrophils. They include N-acetyl tetra, hepta, octa, nona and larger size peptides. Further, it was also revealed that when calpain was incubated with high molecular weight (HMW) or low molecular weight (LMW) kininogen, kinin liberation occurred with simultaneous inhibition of calpains by kininogens. These data suggest that chemical mediators generated from the calpain-kininogen system may participate in migration and accumulation of neutrophils to the inflammatory locus.     

Crystal structure and amide H/D exchange of binary complexes of alcohol dehydrogenase from Bacillus stearothermophilus: insight into thermostability and cofactor binding

The crystal structure of NAD(+)-dependent alcohol dehydrogenase from Bacillus stearothermophilus strain LLD-R (htADH) was determined using X-ray diffraction data at a resolution of 2.35 A. The structure of homotetrameric htADH is highly homologous to those of bacterial and archaeal homotetrameric alcohol dehydrogenases (ADHs) and also to the mammalian dimeric ADHs. There is one catalytic zinc atom and one structural zinc atom per enzyme subunit. The enzyme was crystallized as a binary complex lacking the nicotinamide adenine dinucleotide (NAD(+)) cofactor but including a zinc-coordinated substrate analogue trifluoroethanol. The binary complex structure is in an open conformation similar to ADH structures without the bound cofactor. Features important for the thermostability of htADH are suggested by a comparison with a homologous mesophilic enzyme (55% identity), NAD(+)-dependent alcohol dehydrogenase from Escherichia coli. To gain insight into the conformational change triggered by NAD(+) binding, amide hydrogen-deuterium exchange of htADH, in the presence and absence of NAD(+), was studied by HPLC-coupled electrospray mass spectrometry. When the deuteron incorporation of the protein-derived peptides was analyzed, it was found that 9 of 21 peptides show some decrease in the level of deuteron incorporation upon NAD(+) binding, and another 4 peptides display slower exchange rates. With one exception (peptide number 8), none of the peptides that are altered by bound NAD(+) are in contact with the alcohol-substrate-binding pocket. Furthermore, peptides 5 and 8, which are located outside the NAD(+)-binding pocket, are notable by displaying changes upon NAD(+) binding. This suggests that the transition from the open to the closed conformation caused by cofactor binding has some long-range effects on the protein structure and dynamics.     

Identification of catalytic nucleophile of Escherichia coli gamma-glutamyltranspeptidase by gamma-monofluorophosphono derivative of glutamic acid: N-terminal thr-391 in small subunit is the nucleophile

gamma-Glutamyltranspeptidase (EC 2.3.2.2) is the enzyme involved in glutathione metabolism and catalyzes the hydrolysis and transpeptidation of gamma-glutamyl compounds such as glutathione and its derivatives. The reaction is thought to proceed via a gamma-glutamyl-enzyme intermediate where a hitherto unknown catalytic nucleophile is gamma-glutamylated. Neither affinity labeling nor site-directed mutagenesis of conserved amino acids has succeeded so far in identifying the catalytic nucleophile. We describe here the identification of the catalytic nucleophile of Escherichia coli gamma-glutamyltranspeptidase by a novel mechanism-based affinity labeling agent, 2-amino-4-(fluorophosphono)butanoic acid (1), a gamma-phosphonic acid monofluoride derivative of glutamic acid. Compound 1 rapidly inactivated the enzyme in a time-dependent manner (k(on) = 4.83 x 10(4) M(-1) s(-1)). The inactivation rate was decreased by increasing the concentration of the substrate. The inactivated enzyme did not regain its activity after prolonged dialysis, suggesting that 1 served as an active-site-directed affinity label by phosphonylating the putative catalytic nucleophile. Ion-spray mass spectrometric analyses revealed that one molecule of 1 phosphonylated one molecule of the small subunit. LC/MS experiments of the proteolytic digests of the phosphonylated small subunit identified the N-terminal peptide Thr391-Lys399 as the phosphonylation site. Subsequent MS/MS experiments of this peptide revealed that the phosphonylated residue was Thr-391, the N-terminal residue of the small subunit. We conclude that the N-terminal Thr-391 is the catalytic nucleophile of E. coli gamma-glutamyltranspeptidase. This result strongly suggests that gamma-glutamyltranspeptidase is a new member of the N-terminal nucleophile hydrolase family.     

Kinetics of Acrylodan-Labelled cAMP-Dependent Protein Kinase Catalytic Subunit Denaturation

Fluorescence spectroscopy was used to study denaturation of cAMP-dependent protein kinase catalytic subunit labeled with an acrylodan moiety. The dye was covalently bound to a cystein residue introduced into the enzyme by replacement of arginine in position 326 in the native sequence, located near the enzyme active center. This labeling had no effect on catalytic activity of the enzyme, but provided possibility to monitor changes in protein structure through measuring the fluorescence spectrum of the dye, which is sensitive to changes in its environment. This method was used to monitor denaturation of the protein kinase catalytic subunit and study the kinetics of this process as well as influence of specific ligands on stability of the protein. Stabilization of the enzyme structure was observed in the presence of adenosine triphosphate, peptide substrate RRYSV and inhibitor peptide PKI[5-24].     

Affinity cleavage at the divalent metal site of porcine NAD-specific isocitrate dehydrogenase

A divalent metal ion, such as Mn2+, is required for the catalytic reaction and allosteric regulation of pig heart NAD-dependent isocitrate dehydrogenase. The enzyme is irreversibly inactivated and cleaved by Fe2+ in the presence of O2 and ascorbate at pH 7.0. Mn2+ prevents both inactivation and cleavage. Nucleotide ligands, such as NAD, NADPH, and ADP, neither prevent nor promote inactivation or cleavage of the enzyme by Fe2+. The NAD-specific isocitrate dehydrogenase is composed of three distinct subunits in the ratio 2alpha:1beta:1gamma. The results indicate that the oxidative inactivation and cleavage are specific and involve the 40 kDa alpha subunit of the enzyme. A pair of major peptides is generated during Fe2+ inactivation: 29.5 + 10.5 kDa, as determined by SDS-PAGE. Amino-terminal sequencing reveals that these peptides arise by cleavage of the Val262-His263 bond of the alpha subunit. No fragments are produced when enzyme is incubated with Fe2+ and ascorbate under denaturing conditions in the presence of 6 M urea, indicating that the native structure is required for the specific cleavage. These results suggest that His263 of the alpha subunit may be a ligand of the divalent metal ion needed for the reaction catalyzed by isocitrate dehydrogenase. Isocitrate enhances the inactivation of enzyme caused by Fe2+ in the presence of oxygen, but prevents the cleavage, suggesting that inactivation occurs by a different mechanism when metal ion is bound to the enzyme in the presence of isocitrate: oxidation of cysteine may be responsible for the rapid inactivation in this case. Affinity cleavage caused by Fe2+ implicates alpha as the catalytic subunit of the multisubunit porcine NAD-dependent isocitrate dehydrogenase.     

Protection of tryptic-sensitive sites in the large subunit of ribulosebisphosphate carboxylase/oxygenase by catalysis

Limited tryptic proteolysis of spinach (Spinacia oleracea) ribulose bisphosphate carboxylase/oxygenase (ribulose-P₂ carboxylase) resulted in the ordered release of two adjacent N-terminal peptides from the large subunit, and an irreversible, partial inactivation of catalysis. The two peptides were identified as the N-terminal tryptic peptide (acetylated Pro-3 to Lys-8) and the penultimate tryptic peptide (Ala-9 to Lys-14). Kinetic comparison of hydrolysis at Lys-8 and Lys-14, enzyme inactivation, and changes in the molecular weight of the large subunit, indicated that proteolysis at Lys-14 correlated with inactivation, while proteolysis at Lys-8 occurred much more rapidly. Thus, enzyme inactivation is primarily the result of proteolysis at Lys-14. Proteolysis of ribulose-P₂ carboxylase under catalytic conditions (in the presence of CO₂, Mg²⁺, and ribulose-P₂) also resulted in ordered release of these tryptic peptides; however, the rate of proteolysis at lysyl residues 8 and 14 was reduced to approximately one-third of the rate of proteolysis of these lysyl residues under noncatalytic conditions (in the presence of CO₂ and Mg²⁺ only). The protection of these lysyl residues from proteolysis under catalytic conditions could reflect conformational changes in the N-terminal domain of the large subunit which occur during the catalytic cycle.     

Formate dehydrogenase. Subunit and mechanism of inhibition by cyanide and azide.

Formate dehydrogenase (EC 1.2.1.2) prepared from peas (Pisum sativum) was a two-subunit enzyme. The enzyme accelerated the formation of an NAD+-cyanide compound having an adsorption band at 330 nm. The enzyme was able to bind one NAD+ molecule per each subunit but only 1 mole of NAD+-cyanide compound was formed per two subunits. The complex of NAD+, cyanide, and the enzyme was very stable and had no catalytic activity. Azide inhibited the formate dehydrogenase reaction in two different ways. By incubation of the enzyme with azide in the presence of NAD+, half of its catalytic activity was lost. The remaining activity was also inhibited by azide but this inhibition was removed competively by formate. Contrary to the case of cyanide the inhibition by azide could be removed by dialysis and no spectral species due to the addition compound of NAD+ and azide could be observed. The data from double recipricol plots of the initial velocity and the formate concentration led to a conclusion that formate dehydrogenase has two sites with about equal catalytic activity. The Km for formate was different for the two catalytic sites (1.67 and 6.25 mM) but the difference was not noticeable in the case of the Km for NAD+.