Protein prenyltransferases.
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Alkyl and Aryl Transferases↗
Biomedical subjects
M H Gelb
Publications and source records attributed to M H Gelb.
Structures of free and inhibited human secretory phospholipase A2 from inflammatory exudate.
Phospholipase A2 (PLA2) participates in a wide range of cellular processes including inflammation and transmembrane signaling. A human nonpancreatic secretory PLA2 (hnps-PLA2) has been identified that is found in high concentrations in the synovial fluid of patients with rheumatoid arthritis and in the plasma of patients with septic shock. This enzyme is secreted from certain cell types in response to the proinflammatory cytokines, tumor necrosis factor or interleukin-1. The crystal structures of the calcium-bound form of this enzyme have been determined at physiological pH both in the presence [2.1 angstrom (A) resolution] and absence (2.2 A resolution) of a transition-state analogue. Although the critical features that suggest the chemistry of catalysis are identical to those inferred from the crystal structures of other extracellular PLA2s, the shape of the hydrophobic channel of hnps-PLA2 is uniquely modulated by substrate binding.
Amino Acid Sequence↗
Kinetic characterization of phospholipase A2 modified by manoalogue.
Manoalogue, a synthetic analogue of the sea sponge-derived manoalide, has been previously shown to partially inactivate the phospholipase A2 from cobra venom (Reynolds, L. J., Morgan, B. P., Hite, E. D., Mihelich, E. D., & Dennis, E. A. (1988) J. Am. Chem. Soc. 110, 5172) by reacting with enzyme lysine residues. In the present study, the inactivation of the phospholipases A2 from pig pancreas, bee venom, and cobra (Naja naja naja) venom by manoalogue was studied in detail. Manoalogue-treated enzymes were examined in the scooting mode on vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol. Here the native enzymes bound irreversibly to the vesicles and hydrolyzed all of the phospholipids in the outer monolayer without leaving the surface of the interface. All three manoalogue-treated enzymes showed reduced catalytic turnover for substrate hydrolysis in the scooting mode, and the modified enzymes did not hop from one vesicle to another. Thus, inactivation by manoalogue is not due to the decrease in the fraction of enzyme bound to the substrate interface. This result was also confirmed by fluorescence studies that directly monitored the binding of phospholipase A2 to vesicles. A chemically modified form of the pig pancreatic phospholipase A2 in which all of the lysine epsilon-amino groups have been amidinated was not inactivated by manoalogue, indicating that the modification of lysine residues and not the amino-terminus is required for the inactivation. Several studies indicated that the manoalogue-modified enzymes contain a functional active site. For example, studies that monitored the protection by ligands of the active site from attack by a alkylating agent showed that manoalogue-modified pig phospholipase A2 was capable of binding calcium, a substrate analogue, lipolysis products, and a competitive inhibitor. Furthermore, relative to native enzymes, manoalogue-modified enzymes retained significantly higher catalytic activities when acting on water-soluble substrates than when acting on vesicles in the scooting mode. Intact manoalogue had no affinity for the catalytic site on the enzyme as it did not inhibit the enzyme in the scooting mode and it did not protect the active site from alkylation. Pig pancreatic phospholipase A2 bound to micelles of 2-hexadecyl-sn-glycero-3-phosphocholine was resistant to inactivation by manoalogue, suggesting that the modification of lysine residues on the interfacial recognition surface of the enzyme was required for inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Interfacial catalysis by phospholipase A2: evaluation of the interfacial rate constants by steady-state isotope effect studies.
The kinetics of hydrolysis of phospholipid vesicles by phospholipase A2 (PLA2) in the scooting mode can be described by the Michaelis-Menten formalism for the action of the enzyme in the interface (E*). E* + S in equilibrium E*S in equilibrium E*P in equilibrium E* + Products The values of the interfacial rate constants cannot be obtained by classical methods because the concentration of the substrate within the lipid bilayer is not easily manipulated. In the present study, carbonyl-carbon heavy atom isotope effects for the hydrolysis of phospholipids have been measured in both vesicles and in mixed micelles in which the phospholipid was present in the nonionic detergent Triton X-100. A large [14C]carbonyl carbon isotope effect of 1.12 +/- 0.02 was measured for the cobra venom PLA2-catalyzed hydrolysis of dipalmitoylphosphatidylcholine in Triton X-100. In contrast, no isotope effect (1.01 +/- 0.01) was measured for the action of the porcine pancreatic and cobra venom enzymes on vesicles of dimyristoylphosphatidylmethanol in the scooting mode. In a second experiment, the hydrolysis of vesicles was carried out in oxygen-18 enriched water. Analysis of the released fatty acid product by mass spectrometry showed that it contained only a single oxygen-18. All of these results were used to estimate both the forward and reverse commitments to catalysis. The lack of doubly labeled fatty acid demonstrated that the product is released from the E*P complex faster than the reverse of the esterolysis step. The small isotope effect in vesicles demonstrated that the E*S complex goes on to products faster than substrate is released from the enzyme. The relevance of these results to an understanding of substrate specificity and inhibition of PLA2 is discussed. In addition, the conditions placed on the values of the rate constants obtained in the present study together with results obtained in the other studies described in this series of papers have led to the evaluation of most of the interfacial rate constants for the hydrolysis of phospholipid vesicles by PLA2.
Animals↗
Interfacial catalysis by phospholipase A2: substrate specificity in vesicles.
The binding equilibrium of phospholipase A2 (PLA2) to the substrate interface influences many aspects of the overall kinetics of interfacial catalysis by this enzyme. For example, the interpretation of kinetic data on substrate specificity was difficult when there was a significant kinetic contribution from the interfacial binding step to the steady-state catalytic turnover. This problem was commonly encountered with vesicles of zwitterionic phospholipids, where the binding of PLA2 to the interface was relatively poor. The action of PLA2 on phosphatidylcholine (PC) vesicles containing a small amount of anionic phospholipid, such as phosphatidic acid (PA), was studied. It was shown that the hydrolysis of these mixed lipid vesicles occurs in the scooting mode in which the enzyme remains tightly bound to the interface and only the substrate molecules present on the outer monolayer of the target vesicle became hydrolyzed Thus the phenomenon of scooting mode hydrolysis was not restricted to the action of PLA2 on vesicles of pure anionic phospholipids, but it was also observed with vesicles of zwitterionic lipids as long as a critical amount of anionic compound was present. Under such conditions, the initial rate of hydrolysis of PC in the mixed PC/PA vesicles was enhanced more than 50-fold. Binding studies of PLA2 to vesicles and kinetic studies in the scooting mode demonstrated that the enhancement of PC hydrolysis in the PC/PA covesicles was due to the much higher affinity of the enzyme toward covesicles compared to vesicles of pure PC phospholipids. A novel and technically simple protocol for accurate determination of the substrate specificity of PLA2 at the interface was also developed by using a double-radiolabel approach. Here, the action of PLA2 in the scooting mode was studied on vesicles of the anionic phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphomethanol that contained small amounts of 3H- and 14C-labeled phospholipids. From an analysis of the 3H and 14C radioactivity in the released fatty acid products, the ratio of substrate specificity constants (kcat/KMS) was obtained for any pair of radiolabeled substrates. These studies showed that the PLA2s from pig pancreas and Naja naja naja venom did not discriminate between phosphatidylcholine and phosphatidylethanolamine phospholipids or between phospholipids with saturated versus unsaturated acyl chains and that the pig enzyme had a slight preference for anionic phospholipids (2-3-fold). The described protocol provided an accurate measure of the substrate specificity of PLA2 without complications arising from the differences in binding affinities of the enzyme to vesicles composed of pure phospholipids.
Animals↗
Interfacial catalysis by phospholipase A2: activation by substrate replenishment.
Polymyxin B (Px), a cyclic cationic peptide, was shown to act as a potent activator of interfacial catalysis by phospholipase A2 (PLA2) acting on dimyristoylphosphatidylmethanol vesicles in the scooting mode. A 7-fold increase in the initial enzymatic velocity was seen with the pig pancreatic PLA2 in the presence of 1 microM Px. Initial experiments including the dependency of the degree of activation by Px on the source of the PLA2 suggested that Px bound to a cationic binding site on the enzyme. However, numerous additional observations led to the conclusion that activation by Px was due to its effects on the substrate interface. For example, the activation by Px was only seen when the PLA2 acted on small vesicles rather than larger ones, and all of the available substrate was eventually hydrolyzed in the presence of a small mole fraction of Px. Px did not promote the intervesicle exchange of PLA2, and it did not alter the binding of the evidence led to the conclusion that Px activated interfacial catalysis by promoting the replenishment of substrate in the enzyme-containing vesicles. When PLA2 was acting on small vesicles in the scooting mode, the observed initial velocity was lower than that measured with large vesicles because the surface concentration of substrate decreased relatively rapidly in the small vesicles. Px promoted the transfer of phospholipids between the vesicles and functioned as an activator by keeping the mole fraction of substrate in the enzyme-containing vesicles close to 1. This effect of Px was consistent with the ability of polycationic peptides to induce the intervesicle mixing of anionic phospholipids in vesicles [Bondeson, J., & Sundler, R. (1990) Biochim. Biophys. Act 1026, 186-194]. Activation by substrate replenishment was quantitatively predicted by the theory of interfacial catalysis on vesicles in the scooting mode. The role of substrate replenishment in the kinetics of interfacial catalysis in phospholipid micelles was discussed. Finally, the protocols developed in this paper were outlined in view of their utility in the analysis of activators of interfacial catalysis.
Amino Acid Sequence↗
C terminus of the small GTP-binding protein smg p25A contains two geranylgeranylated cysteine residues and a methyl ester.
smg p25A, also known as the rab3A protein, is a small GTP-binding protein that has been implicated in intracellular vesicle transport and the secretion of neurotransmitters. It has been shown to bind reversibly to membranes, though its cDNA-predicted sequence contains no obvious membrane-binding domains. However, smg p25A does contain a cDNA-predicted C-terminal Cys-Ala-Cys sequence at positions 218 through 220, which suggests that it may be posttranslationally modified. In the present study we used two different approaches to investigate this possibility. First, we incubated pheochromocytoma cells with [3H]mevalonolactone, examined the proteins that became labeled by two-dimensional gel electrophoresis, and demonstrated that two of these proteins exactly corresponded to smg p25A. Second, we purified smg p25A from bovine brain membranes and analyzed both the full-length protein and a proteolytically derived C-terminal peptide by a combination of high performance liquid chromatography and mass spectrometry. This approach revealed that the protein's C-terminal region is methyl-esterified and contains two geranylgeranyl groups linked via thioether bonds to Cys-218 and Cys-220. Since smg p25A is one of several small GTP-binding proteins that share a C-terminal Cys-Xaa-Cys consensus sequence (where Xaa is an unspecified amino acid), our results suggest that these proteins may be similarly geranylgeranylated and methyl-esterified.
Adrenal Gland Neoplasms↗
A protein geranylgeranyltransferase from bovine brain: implications for protein prenylation specificity.
A protein geranylgeranyltransferase (PGT) that catalyzes the transfer of a 20-carbon prenyl group from geranylgeranyl pyrophosphate to a cysteine residue in protein and peptide acceptors was detected in bovine brain cytosol and partially purified. The enzyme was shown to be distinct from a previously characterized protein farnesyltransferase (PFT). The PGT selectively geranylgeranylated a synthetic peptide corresponding to the C terminus of the gamma 6 subunit of bovine brain G proteins, which have previously been shown to contain a 20-carbon prenyl modification. Likewise, a peptide corresponding to the C terminus of human lamin B, a known farnesylated protein, selectively served as a substrate for farnesylation by the PFT. However, with high concentrations of peptide acceptors, both prenyl transferases were able to use either peptide as substrates and the PGT was able to catalyze farnesyl transfer. Among the prenyl acceptors tested, peptides and proteins with leucine or phenylalanine at their C termini served as geranylgeranyl acceptors, whereas those with C-terminal serine were preferentially farnesylated. These results suggest that the C-terminal amino acid is an important structural determinant in controlling the specificity of protein prenylation.
Alkyl and Aryl Transferases↗
Protein geranylgeranyltransferase of Saccharomyces cerevisiae is specific for Cys-Xaa-Xaa-Leu motif proteins and requires the CDC43 gene product but not the DPR1 gene product.
Protein prenylation occurs by modification of proteins with one of at least two isoprenoids, the farnesyl group and the geranylgeranyl group. Protein farnesyltransferases have been identified, but no such enzyme has been identified for geranylgeranylation. We report the identification of an activity in crude soluble yeast extracts that catalyzes the transfer of a geranylgeranyl moiety from geranylgeranyl pyrophosphate to proteins having the C-terminal sequence Cys-Ile-Ile-Leu or Cys-Val-Leu-Leu but not to a similar protein ending with Cys-Ile-Ile-Ser. This activity is dependent upon the CDC43/CAL1 gene, which is involved in budding and the control of cell polarity, but does not require the DPR1/RAM1 gene, which is known to be required for the farnesylation of Ras proteins. These results indicate that the protein geranylgeranyltransferase activity is distinct from the protein farnesyltransferase activity and that its specificity depends in part on the extreme C-terminal leucine in the protein to be prenylated.
Alkyl and Aryl Transferases↗
Crystal structure of bee-venom phospholipase A2: correction.
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Amino Acid Sequence↗
Membrane-binding domain of the small G protein G25K contains an S-(all-trans-geranylgeranyl)cysteine methyl ester at its carboxyl terminus.
We showed previously that a 23-kDa guanine nucleotide-binding protein (G protein) purified from bovine brain membranes is carboxyl methylated and that this modification occurs at or near the membrane-binding domain. In the present study, we identified this small G protein as G25K (formerly termed Gp). We demonstrated that proteolytic digests of 3H-methylated G25K contained radiolabeled material that coeluted with synthetic S-(geranylgeranyl)cysteine methyl ester on reversed-phase HPLC. Further treatment by performic acid oxidation yielded radiolabeled material that coeluted with L-cysteic acid methyl ester, verifying that the isoprenoid moiety and carboxyl methyl ester are localized on a C-terminal cysteine residue. Analysis by gas chromatography-coupled mass spectrometry of material released from purified G25K by Raney nickel treatment positively identified the covalently bound lipid as an all-trans-geranylgeranyl (C20) isoprenoid moiety. These results suggest that geranylgeranyl modification and perhaps methyl esterification function in the membrane localization of this small G protein.
Amino Acid Sequence↗
Phospholipase A2-catalyzed hydrolysis of vesicles: uses of interfacial catalysis in the scooting mode.
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Catalysis↗
Interfacial catalysis: the mechanism of phospholipase A2.
A chemical description of the action of phospholipase A2 (PLA2) can now be inferred with confidence from three high-resolution x-ray crystal structures. The first is the structure of the PLA2 from the venom of the Chinese cobra (Naja naja atra) in a complex with a phosphonate transition-state analogue. This enzyme is typical of a large, well-studied homologous family of PLA2S. The second is a similar complex with the evolutionarily distant bee-venom PLA2. The third structure is the uninhibited PLA2 from Chinese cobra venom. Despite the different molecular architectures of the cobra and bee-venom PLA2s, the transition-state analogue interacts in a nearly identical way with the catalytic machinery of both enzymes. The disposition of the fatty-acid side chains suggests a common access route of the substrate from its position in the lipid aggregate to its productive interaction with the active site. Comparison of the cobra-venom complex with the uninhibited enzyme indicates that optimal binding and catalysis at the lipid-water interface is due to facilitated substrate diffusion from the interfacial binding surface to the catalytic site rather than an allosteric change in the enzyme's structure. However, a second bound calcium ion changes its position upon the binding of the transition-state analogue, suggesting a mechanism for augmenting the critical electrophile.
Bee Venoms↗
Crystal structure of cobra-venom phospholipase A2 in a complex with a transition-state analogue.
The crystal structure of a complex between a phosphonate transition-state analogue and the phospholipase A2 (PLA2) from Naja naja atra venom has been solved and refined to a resolution of 2.0 angstroms. The identical stereochemistry of the two complexes that comprise the crystal's asymmetric unit indicates both the manner in which the transition state is stabilized and how the hydrophobic fatty acyl chains of the substrate are accommodated by the enzyme during interfacial catalysis. The critical features that suggest the chemistry of binding and catalysis are the same as those seen in the crystal structure of a similar complex formed with the evolutionarily distant bee-venom PLA2.
Amino Acid Sequence↗
Crystal structure of bee-venom phospholipase A2 in a complex with a transition-state analogue.
The 2.0 angstroms crystal structure of a complex containing bee-venom phospholipase A2 (PLA2) and a phosphonate transition-state analogue was solved by multiple isomorphous replacement. The electron-density map is sufficiently detailed to visualize the proximal sugars of the enzyme's N-linked carbohydrate and a single molecule of the transition-state analogue bound ot its active center. Although bee-venom PLA2 does not belong to the large homologous Class I/II family that encompasses most other well-studied PLA2s, there is segmental sequence similarity and conservation of many functional substructures. Comparison of the bee-venom enzyme with other phospholipase structures provides compelling evidence for a common catalytic mechanism.
Amino Acid Sequence↗
Kinetic and inhibition studies of phospholipase A2 with short-chain substrates and inhibitors.
The action of the phospholipases A2 (PLA2s) from Naja naja naja, Naja naja atra, and Crotalus atrox venoms as well as the enzyme from porcine pancreas on a number of short-chain, water-soluble substrates was studied. The inhibition of these enzymes by short-chain phosphonate- and thiophosphonate-containing phospholipid analogues was also examined. The kinetic patterns observed for the action of the venom PLA2s on substrates containing phosphocholine head groups all deviated from a classical Michaelis-Menten-type behavior. With a substrate containing an anionic head group, the kinetic pattern observed was more normal. In contrast, Michaelis-Menten-type behavior was observed for the action of the porcine pancreatic PLA2 acting on all of the substrates studied. A short-chain phospholipid analogue in which the enzyme-susceptible ester was replaced with a phosphonate group was found to be a tight-binding inhibitor of the venom PLA2s with IC50 values that were some 10(4)-10(5)-fold lower than the concentration of substrate used in the assay. The degree of inhibition was found to depend dramatically on the stereochemical arrangement of substituents in the inhibitor which strongly suggests that the inhibitors are binding directly to the active site of the PLA2s. By comparison, the phosphonate analogue functioned as a poor inhibitor of the porcine pancreatic PLA2. Direct inhibitor binding studies indicated that the short-chain phosphonate inhibitor bound weakly to the venom enzymes in the absence of the short-chain substrates. Several other unusual features of the inhibition were also observed. The data are interpreted in terms of a model in which the enzyme and substrate form a lipid-protein aggregate at substrate concentrations below the critical micelle concentration (cmc). Possible reasons for the selective binding of the inhibitor to the enzyme-substrate microaggregate are discussed.
Animals↗
Identification of geranylgeranyl-modified proteins in HeLa cells.
Previous studies have shown that animal cells contain isoprenoid-modified proteins and that one of these proteins, lamin B, contains a thioether-linked farnesyl group that is attached to cysteine. In the present study, a novel isoprenoid-modification was identified by labeling HeLa cells with [3H]mevalonic acid and analyzing proteolytic digests of the total cell protein. Radioactive fragments were purified from these digests and treated with Raney nickel. The released, labeled material was analyzed by gas-liquid chromatography (GC) and mass spectrometry (MS). This approach revealed that an all-trans geranylgeranyl group was a major isoprenoid modification.
Chromatography, Gel↗
Prenyl proteins in eukaryotic cells: a new type of membrane anchor.
Recent studies have indicated that eukaryotic cells contain proteins that are post-translationally modified by long-chain, thioether-linked prenyl groups. These proteins include yeast mating factors, ras proteins and nuclear lamins. The modification occurs on a cysteine residue near the C terminus and appears to initiate a set of additional protein modification reactions that promote attachment of the proteins to specific membranes.
Animals↗