The active-site environment of rhodopsin

The 11-cis-retinal binding site of rhodopsin is of great interest because it is buried in the membrane but yet must provide an environment for charged amino acids. In addition, the active-site lysine residue must be able to engage in rapid Schiff base formation with 11-cis-retinal at neutral and lower pH values. This requires that this lysine be unprotonated. We have begun to study the environment of the active-site lysine using a reporter group adducted to it. Non-active-site permethylated opsin was reacted with 5-nitrosalicylaldehyde, and the resulting Schiff base was permanently fixed by borohydride reduction. The stoichiometry of incorporation was one. This chromophoric and pH-sensitive reporter group affords information on the active-site environment of rhodopsin by determining the ionization constants of its ionizable groups at different pH values. The pH titration of the modified protein showed a single pKa = 7.8 +/- 0.19 ascribable to the ionization of the phenol. The ionization of the modified lysine residue was not observed at all pH values studied. These studies are interpreted to mean that a negatively charged amino acid is propinquous to the active-site lysine residue and that this latter residue does not have an unusually low pKa.     

An active-site lysine in avian liver phosphoenolpyruvate carboxykinase

The participation of lysine in the catalysis by avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification and by a characterization of the modified enzyme. The rate of inactivation by 2,4-pentanedione is pseudo-first-order and linearly dependent on reagent concentration with a second-order rate constant of 0.36 +/- 0.025 M-1 min-1. Inactivation by pyridoxal 5'-phosphate of the reversible reaction catalyzed by phosphoenolpyruvate carboxykinase follows bimolecular kinetics with a second-order rate constant of 7700 +/- 860 M-1 min-1. A second-order rate constant of inactivation for the irreversible reaction catalyzed by the enzyme is 1434 +/- 110 M-1 min-1. Treatment of the enzyme with pyridoxal 5'-phosphate gives incorporation of 1 mol of pyridoxal 5'-phosphate per mole of enzyme or one lysine residue modified concomitant with 100% loss in activity. A stoichiometry of 1:1 is observed when either the reversible or the irreversible reactions catalyzed by the enzyme are monitored. A study of kobs vs pH suggests this active-site lysine has a pKa of 8.1 and a pH-independent rate constant of inactivation of 47,700 M-1 min-1. The phosphate-containing substrates IDP, ITP, and phosphoenolpyruvate offer almost complete protection against inactivation by pyridoxal 5'-phosphate. Modified, inactive enzyme exhibits little change in Mn2+ binding as shown by EPR. Proton relaxation rate measurements suggest that pyridoxal 5'-phosphate modification alters binding of the phosphate-containing substrates. 31P NMR relaxation rate measurements show altered binding of the substrates in the ternary enzyme.Mn2+.substrate complex. Circular dichroism studies show little change in secondary structure of pyridoxal 5'-phosphate modified phosphoenolpyruvate carboxykinase. These results indicate that avian liver phosphoenolpyruvate carboxykinase has one reactive lysine at the active site and it is involved in the binding and activation of the phosphate-containing substrates.     

The incorporation of tritiated retinyl moiety into the active-site lysine residue of bacteriorhodopsin.

Purple membranes were isolated from Halobacterium halobium bleached and regenerated with all-trans-[15-3H]retinal. The incorporation of label was 1.2 mol of retinal/mol of bacterio-opsin. The [3H]retinyl-bacterio-opsin obtained from regeneration was hydrolysed to give tritiated retinyl-lysine, which, on hydrogenation to N-epsilon-perhydro[3H]retinyl-lysine and reaction with 1-fluoro-2,4-dinitrobenzene, gave bis-(2,4-dinitrophenyl)-N-epsilon-perhydro[3H]retinyl-lysine. This result confirmed that the retinyl moiety of the chromophore is attached to an epsilon-amino group of lysine.     

Chemical modification of lysine residues at active-site of human placental estradiol 17 beta-dehydrogenase

Estradiol 17 beta-dehydrogenase (EC 1.1.1.62.) activity was decreased by 2,4,6-trinitrobenzene sulfonate (TNBS), a reagent for modification of epsilon-amino moiety of lysine residues in a protein. The inactivation exhibited pseudo-first-order kinetics, and was protected by oxidyzed cofactors. Stoichiometric studies showed that the complete inactivation was caused by modification of one lysine residue per molecule of the enzyme. Differential modification with 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB), TNBS and dithiothreitol (DTT) indicated that the residues of lysine and cysteine were located at the active-site and played an essential role in the catalytic function of the estradiol 17 beta-dehydrogenase.     

Characterization of the active-site residues asparagine 167 and lysine 161 of the IMP-1 metallo beta-lactamase

The roles of lysine at position 161 and asparagine at position 167 in IMP-1 metallo beta-lactamase were studied by site-directed mutagenesis. These residues are highly conserved in metallo beta-lactamases and are thought to be present in the active-site cavity. Mutant enzymes with alanine or aspartic acid at position 167 showed almost the same properties as the wild-type enzyme. Kinetic parameters for the mutant enzymes differing at position 161 indicated that the positive charge of lysine 161 is required for electrostatic interaction with the carboxyl moiety of the substrate, i.e. C-3 of penicillins or C-4 of cephalosporins.     

Methylation of histone H3 lysine 9 occurs during translation

Histone post-translational modifications are key contributors to chromatin structure and function, and participate in the maintenance of genome stability. Understanding the establishment and maintenance of these marks, along with their misregulation in pathologies is thus a major focus in the field. While we have learned a great deal about the enzymes regulating histone modifications on nucleosomal histones, much less is known about the mechanisms establishing modifications on soluble newly synthesized histones. This includes methylation of lysine 9 on histone H3 (H3K9), a mark that primes the formation of heterochromatin, a critical chromatin landmark for genome stability. Here, we report that H3K9 mono- and dimethylation is imposed during translation by the methyltransferase SetDB1. We discuss the importance of these results in the context of heterochromatin establishment and maintenance and new therapeutic opportunities in pathologies where heterochromatin is perturbed.     

Substitution of S-(beta-aminoethyl)-cysteine for active-site lysine of thermostable aspartate aminotransferase

The active site lysyl residue (K239) of the thermostable aspartate aminotransferase [EC 2.6.1.1] was replaced by cysteinyl residue by means of site-directed mutagenesis. The K239C mutant enzyme obtained was catalytically inactive. The reaction of the cysteinyl residue of the K239C mutant enzyme with ethylenimine led to the formation of S-(beta-aminoethylcysteinyl (SAEC) residue. The K239SAEC mutant enzyme obtained showed about 25% of the activity of wild-type enzyme, and absorbed at 375 nm, which suggested the internal Schiff base formation.     

Methylation of lysine residues of histone fractions in synchronized mommalian cells

Synchronized cultures of mammalian cells were labeled with ¹⁴C-methyl methionine. Labeled methionine methyl groups were incorporated into certain histone fractions, forming methyl lysine. Incorporation of labeled methyl group into histone fractions as ¹⁴C-methyl lysine was followed through the cell cycle from late G₁ into early M. The ¹⁴C-methyl lysine contents of fractions F2a and F3 began to rise in S and reached maxima after termination of DNA and histone synthesis, coincident with the beginning of mitosis, and began to fall by mid-M. The ¹⁴C-methyl lysine content of fraction F2b rose to a maximum early in S, coincident with initiation of DNA synthesis, and rapidly decreased to its original unmethylated level by late S. Fraction F1 remained unmethylated during the period G₁-M. Evidence is presented to demonstrate differential methylation of histone fractions and to substantiate differential temporal coupling of the methylation of specific histone fractions with histone and DNA biosynthesis.     

His166 is critical for active-site proton transfer and phototaxis signaling by sensory rhodopsin I.

Photoinduced deprotonation of the retinylidene Schiff base in the sensory rhodopsin I transducer (SRI-Htrl) complex results in formation of the phototaxis signaling state S373. Here we report identification of a residue, His166, critical to this process, as well as to reprotonation of the Schiff base during the recovery phase of the SRI photocycle. Each of the residue substitutions A, D, G, L, S, V, or Y at position 166 reduces the flash yield of S373, to values ranging from 2% of wild type for H166Y to 23% for H166V. The yield of S373 is restored to wild-type levels in Htrl-free H166L by alkaline deprotonation of Asp76, a Schiff base proton acceptor normally not ionized in the SRI-Htrl complex, showing that proton transfer from the Schiff base in H166L occurs when an acceptor is made available. The flash yield and rate of decay of S373 of the mutants are pH dependent, even when complexed with Htrl, which confers pH insensitivity to wild-type SRI, suggesting that partial disruption of the complex has occurred. The rates of S373 reprotonation at neutral pH are also prolonged in all H166X mutants, with half-times from 5 s to 160 s (wild type, 1 s). All mutations of His166 tested disrupt phototaxis signaling. No response (H166D, H166L), dramatically reduced responses (H166V), or inverted responses to orange light (H166A, H166G, H166S, and H166Y) or to both orange and near-UV light (H166Y) are observed. Our conclusions are that His166 1) plays a role in the pathways of proton transfer both to and from the Schiff base in the SRI-Htrl complex, either as a structurally important residue or possibly as a participant in proton transfers; 2) is involved in the modulation of SRI photoreaction kinetics by Htrl; and 3) is important in phototaxis signaling. Consistent with the involvement of the His imidazole moiety, the addition of 10 mM imidazole to membrane suspensions containing H166A receptors accelerates S373 decay 10-fold at neutral pH, and a negligible effect is seen on wild-type SRI.     

Leucine dehydrogenase from Bacillus stearothermophilus: identification of active-site lysine by modification with pyridoxal phosphate.

We have constructed an efficient expression plasmid for the leucine dehydrogenase gene previously cloned from Bacillus stearothermophilus. The recombinant enzyme was overproduced in Escherichia coli cells to a level of more than 30% of the total soluble protein upon induction with isopropyl beta-D-thiogalactopyranoside. The enzyme could be readily purified to homogeneity by heat treatment and a single step of ion-exchange chromatography. The purified enzyme was inactivated in a time-dependent manner upon incubation with pyridoxal 5'-phosphate (PLP) followed by reduction with sodium borohydride. The inactivation was completely prevented in the copresence of L-leucine and NAD+. Concomitantly with the inactivation, several molecules of PLP were incorporated into each subunit of the hexameric enzyme. Sequence analysis of the fluorescent peptides isolated from a proteolytic digest of the modified protein revealed that Lys80, Lys91, Lys206, and Lys265 were labeled. Among these residues, Lys80 was predominantly labeled and, in the presence of L-leucine and NAD+, was specifically protected from the labeling. Furthermore, a linear relationship of about 1:1 was observed between the extent of inactivation and the amount of PLP incorporated into Lys80. A slightly active mutant enzyme, in which Lys80 is replaced by Ala, was not inactivated at all by incubation with PLP, showing that the inactivation is correlated with the labeling of only Lys80. Lys80is conserved in the corresponding regions of all the amino acid dehydrogenase sequences reported to date. These results suggest that Lys80 is located at the active site and plays an important role in the catalytic function of leucine dehydrogenase.     

Amino acid sequence of the pyruvate and the glyoxylate active-site lysine peptide of Escherichia coli 2-keto-4-hydroxyglutarate aldolase

Pure 2-keto-4-hydroxyglutarate aldolase of Escherichia coli, a "lysine-type" trimeric enzyme which has the unique properties of forming an "abortive" Schiff-base intermediate with glyoxylate (the aldehydic product/substrate) and of showing strong beta-decarboxylase activity toward oxalacetate, binds any one of its substrates (2-keto-4-hydroxyglutarate, pyruvate, or glyoxylate) in a competitive manner. To determine whether the substrates bind at the same or different (juxta-positioned) sites and what degree of homology might exist between the active-site lysine peptide of this enzyme and that of other lysine-type (Class I) aldolases or beta-decarboxylases, the azomethine formed separately by this aldolase with either [14C]pyruvate or [14C]glyoxylate was reduced with CNBH3-. After each enzyme adduct was digested with trypsin, the 14C-labeled peptide was isolated, purified, and subjected to amino acid analysis and sequence determination. In each case, the same 14-amino acid lysine-peptide was isolated and found to have the following primary sequence: Glu-Phe-*Lys-Phe-Phe-Pro-Ala-Glu-Ala-Asn-Gly-Gly-Val-Lys (where * = the active-site lysine). Hence, glyoxylate competes for, and inhibits aldolase activity by reacting with, the one active-site lysine residue/subunit. This active-site lysine peptide has a high degree (65%) of homology with that of 2-keto-3-deoxy-6-phosphogluconate aldolase of Pseudomonas putida but is not similar to that of any Class I fructose-1,6-bisphosphate aldolase or of acetoacetate beta-decarboxylase of Clostridium acetobutylicum. Furthermore, it was found that extensive reaction of glyoxylate with the N-terminal amino group of this enzyme may well be general complicating factor in sequence studies with proteins plus glyoxylate.     

Methylation changes of lysine 9 of histone H3 during preimplantation mouse development

Immediately after fertilization, a chromatin remodeling process in the oocyte cytoplasm extracts protamine molecules from the sperm-derived DNA and loads histones onto it. We examined how the histone H3-lysine 9 methylation system is established on the remodeled sperm chromatin in mice. We found that the paternal pronucleus was not stained for dimethylated H3-K9 (H3-m2K9) during pronucleus development, while the maternal genome stained intensively. Such H3-m2K9 asymmetry between the parental pronuclei was independent of HP1b localization and, much like DNA methylation, was preserved to the two-cell stage when the nucleus appeared to be compartmentalized for H3-m2K9. A conspicuous increase in H3-m2K9 level was observed at the four-cell stage, and then the level was maintained without a visible change up to the blastocyst stage. The behavior of H3-m2K9 was very similar, but not identical, to that of 5-methylcytosine during preimplantation development, suggesting that there is some connection between methylation of histone and of DNA in early mouse development.     

A single amino acid substitution in rhodopsin (lysine 248----leucine) prevents activation of transducin

In structure-function studies on bovine rhodopsin by in vitro site-specific mutagenesis, we have prepared three mutants in the cytoplasmic loop between the putative transmembrane helices E and F. In each mutant, charged amino acid residues were replaced by neutral residues: mutant 1, Glu239----Gln; mutant 2, Lys248----Leu; and mutant 3, Glu247----Gln, Lys248----Leu, and Glu249----Gln. The mutant rhodopsin genes were expressed in monkey kidney (COS-1) cells. After the addition of 11-cis-retinal to the cells, the rhodopsin mutants were purified by immunoaffinity adsorption. Each mutant gave a wild-type rhodopsin visible absorption spectrum. The mutants were assayed for their ability to stimulate the GTPase activity of transducin in a light-dependent manner. While mutants 1 and 3 showed wild-type activity, mutant 2 (Lys248----Leu) was inactive.     

Methylation of Drosophila histones at proline, lysine, and arginine residues during heat shock

Heat shock or arsenite treatment alter the pattern of histone methylation in Drosophila cells. Both types of stress induce a rapid increase in the methylation level of histone H2B. The methylated amino acid residue of H2B has been identified by thin layer chromatography and electrophoresis as methylproline and is located at the N-terminal end of H2B. Heat shock also induces a decrease in the level of methylation of histone H3. Under normal growth temperature conditions, histone H3 is shown to be methylated on lysine residues. However under heat shock conditions, there is a decrease in the extent of methylation of lysine residues and the appearance of new methylation on arginine residues in H3. These new heat shock-induced methylated residues have been identified as the symmetrical and asymmetrical forms of dimethylarginine. The methylated amino acid residue of histone H4 is lysine with mono-, di-, and trimethyl forms found in both control and heat or chemically stressed cells. These stress-induced changes in the methylation level of the N-terminal proline residue of histone H2B and shift in the methylation sites of histone H3 may be involved in the restructuration of chromatin accompanying the inactivation of normal genes in response to stress. Moreover, we suggest that the hypermethylation of H2B may also be involved in its protection from increased ubiquitin-mediated proteolytic activity under these conditions of cellular stress.     

Methylation of histone H3 lysine 36 is required for normal development in Neurospora crassa

The SET domain is an evolutionarily conserved domain found predominantly in histone methyltransferases (HMTs). The Neurospora crassa genome includes nine SET domain genes (set-1 through set-9) in addition to dim-5, which encodes a histone H3 lysine 9 HMT required for DNA methylation. We demonstrate that Neurospora set-2 encodes a histone H3 lysine 36 (K36) methyltransferase and that it is essential for normal growth and development. We used repeat induced point mutation to make a set-2 mutant (set-2(RIP1)) with multiple nonsense mutations. Western analyses revealed that the mutant lacks SET-2 protein and K36 methylation. An amino-terminal fragment that includes the AWS, SET, and post-SET domains of SET-2 proved sufficient for K36 HMT activity in vitro. Nucleosomes were better substrates than free histones. The set-2(RIP1) mutant grows slowly, conidiates poorly, and is female sterile. Introducing the wild-type gene into the mutant complemented the defects, confirming that they resulted from loss of set-2 function. We replaced the wild-type histone H3 gene (hH3) with an allele producing a Lys to Leu substitution at position 36 and found that this hH3(K36L) mutant phenocopied the set-2(RIP1) mutant, confirming that the observed defects in growth and development result from inability to methylate K36 of H3. Finally, we used chromatin immunoprecipitation to demonstrate that actively transcribed genes in Neurospora crassa are enriched for H3 methylated at lysines 4 and 36. Taken together, our results suggest that methylation of K36 in Neurospora crassa is essential for normal growth and development.     

Methylation of histone H4 lysine 20 controls recruitment of Crb2 to sites of DNA damage

Histone lysine methylation is a key regulator of gene expression and heterochromatin function, but little is known as to how this modification impinges on other chromatin activities. Here we demonstrate that a previously uncharacterized SET domain protein, Set9, is responsible for H4-K20 methylation in the fission yeast Schizosaccharomyces pombe. Surprisingly, H4-K20 methylation does not have any apparent role in the regulation of gene expression or heterochromatin function. Rather, we find the modification has a role in DNA damage response. Loss of Set9 activity or mutation of H4-K20 markedly impairs cell survival after genotoxic challenge and compromises the ability of cells to maintain checkpoint mediated cell cycle arrest. Genetic experiments link Set9 to Crb2, a homolog of the mammalian checkpoint protein 53BP1, and the enzyme is required for Crb2 localization to sites of DNA damage. These results argue that H4-K20 methylation functions as a "histone mark" required for the recruitment of the checkpoint protein Crb2.