Insights into the active site of AMP-forming acetyl-CoA synthetase (2024)

Progress 07/01/02 to 06/30/07

Outputs
Acetyl-CoA synthetase (ACS) catalyzes the formation of acetyl-CoA, a central intermediate in metabolism, from acetate, ATP, and Coenzyme A (CoA). As our model system for investigating the active site of ACS, we have chosen to focus on Methanobacterium thermautotrophicus, which has two ORFs (designated MT-ACS1 and MT-ACS2) with high identity to ACS. MT-ACS1 has been biochemically and kinetically characterized as has an additional ACS (AF-ACS) from the sulfur-reducing archaeon Archaeoglobus fulgidus. The characterization of these ACSs indicates that the ACS family of enzymes shows unexpected substrate diversity. Using the published structure of the Salmonella enterica ACS and modeled structures for MT-ACS1 and AF-ACS, we identified key active site residues and characterized their roles in substrate binding and catalysis. These includes residues for acetate, ATP, and CoA binding and catalysis. We have identified the four residues that comprise the acyl substrate bindingpocket and shown that a conserved tryptophan (Trp-416 of MT-ACS1) is the major determinant for substrate specificity and ranges. A conserved aspartate (Asp-502) was shown to be a key residue for ATP binding and catalysis, and three conserved arginine (Arg-193, Arg-528, and Arg-586 of MT-ACS1) residues were shown to be critical for CoA binding and catalysis. An ACS-like enzyme in M. thermautotrophicus has also been identified and characterized. Surprisingly, when utilizing short chain substrates such as propionate this enzyme lacks the ability to complete the thioester-forming second step of the acyl-CoA synthetase reaction and instead releases the acyl-adenylate product from the first step. In fact, CoA is a non-competitive inhibitor of this reaction. hom*ologs of this enzyme, designated as AAS for acyl-adenylate synthetase, have been identified in the completed genomes of many methanoarchaea and a few anaerobic bacteria. An AAS from Methanosarcina acetivorans has been biochemicallyand kinetically characterized and found to have similar properties to the M. thermautotrophicus AAS. Unexpectedly, AAS has been shown to form CoA thioester products (acyl-CoAs) in the presence of longer or branched chain acyl substrates. For substrates such as methyl-butyrate, AAS forms a methyl-butyryl-CoA in the presence of CoA. Thus, AAS is a bifunctional enzyme in which formation of the acyl-CoA thioester is dependent on the affinity of the acyl substrate in the first step of the reaction. To further investigate the enzymology of AAS, a collaboration has been established with Dr. Andrew Gulick (SUNY-Buffalo) to solve its structure. Recently, the 2.6 angstrom structure of the M. acetivorans AAS was determined. MA-AAS was found to be in a similar conformation to that of the Salmonella enterica ACS in the thioester-forming conformation. This is the first demonstration of an unliganded enzyme from the acyl-adenylate forming superfamily in the thioester-forming conformation, and is incontradiction to the domain alternation hypothesis proposed by Dr. Gulick, raising the possibility that only some enzymes in the superfamily undergo this conformation change between the two steps of the reaction.

Impacts
Acetyl-CoA, an essential intermediate at the junction of various anabolic and catabolic pathways, plays a central role in carbon metabolism in all domains of life. In animals and plants, ACS is the enzyme responsible for the synthesis of acetyl-CoA from acetate. Astonishingly, the regulation of acetate metabolism is conserved from bacteria to mammals. The recent findings that sirtuins regulate ACS activity in humans have implications for pathologies such as diabetes as well as the modulation of lifespan. Thus our research into the mechanism of ACS may eventually have more direct application to human health and well being. I received a grant from National Institutes of Health (GM069374-01A1) to investigate the active site(s) of ACS. A proposal to the National Science Foundation to further our studies on both ACS and AAS is under review. Three papers have been published, one manuscript has been submitted, and four additional manuscripts are in preparation and are nearsubmission. The data generated from this project was presented at the general meeting of the American Society for Microbiology in 2005. I have given invited presentations at the College of Charleston, University of South Carolina, and the University of Georgia. I am also an invited speaker at the prestigious Gordon Conference for Carbon-1 metabolism in the summer of 2008. I have trained number of undergraduate and graduate students have been trained on this project. Jerry Thurman, a minority student, received his MS in Biochemistry and is now enrolled in medical school at the University of South Carolina. Aigerim Bizhanova received her MS in Genetics and is currently enrolled in the PhD program in Human Biology at Northwestern University. Yu Meng is in her third year in the PhD program in Biochemistry and Molecular Biology and recently passed her qualifying exam. Over thirty undergraduates, including twenty biochemistry majors and five genetics majors, eight summer students from theNSF/NIH sponsored Bioengineering and Bioinformatics Summer Institute, and two students from the NSF REU program in the Department of Genetics & Biochemistry, have performed their research in my lab. These students have been accepted into professional and graduate programs at institutions such as Brown, Georgetown, Harvard, Northwestern, Vanderbilt, and Wake Forest, among others. Two undergraduates have been authors on published papers from this lab, and a number of undergraduates are authors on manuscripts that are currently being prepared for submission.

Publications

Progress 01/01/06 to 12/31/06

Outputs
The key enzyme for synthesis of acetyl-CoA from acetate is AMP-forming acetyl-CoA synthetase (ACS). A number of archaea have multiple ORFs with high identity to ACS. MT-ACS1 from the methane-producing archaeon Methanothermobacter thermautotrophicus and AF-ACS2 from the sulfur-reducing archaeon Archaeoglobus fulgidus have been recombinantly produced and purified from Escherichia coli. The biochemical and kinetic characterization of these archaeal enzymes indicates that the ACS family shows unexpected substrate diversity (Archaea 2: 95-107, 2006). Using the published structure of the Salmonella enterica ACS, a number of active site residues for acetate, ATP, and CoA binding and catalysis have been identified. Of the four residues that form the acetate binding pocket of MT-ACS1, we have shown that Trp-416 (and Val-388 to a lesser extent) is a major determinant in acyl substrate utilization (Biochemistry 45:11482-90, 2006). Structural modeling has been performed toidentify additional residues that contribute to acyl substrate choice and preference. Site-directed alteration of these residues and purification and characterization of the enzymes variants is ongoing. In addition, conserved motif III residues in ACS have been shown to play a key role in ATP binding and catalysis (J. Thurman et al., manuscript in preparation). Finally, two conserved Arg residues have been shown to be key residues for CoA binding and catalysis (C. Ingram-Smith et al., manuscript in preparation). We have also begun characterization of a group of short chain acyl-CoA synthetases (designated here as XCSs) for which the substrate specificity is unknown. Our initial characterization indicates that the previously unidentified human XCS may be a propionyl-CoA synthetase. A full biochemical and kinetic characterization of the human XCS is currently being performed. A third ACS-like enzyme in M. thermautotrophicus has been identified and characterized. Unexpectedly, thisenzyme lacks the ability to complete the thioester-forming second step of the acyl-CoA synthetase reaction and instead releases the acyl-adenylate product from the first step. hom*ologs of this enzyme, designated as AAS for acyl-adenylate synthetase, have been identified in the completed genomes of many methanoarchaea and a few anaerobic bacteria. An AAS from methane-producing archaeon Methanosarcina acetivorans has been biochemically and kinetically characterized and found to have similar properties to the M. thermautotrophicus AAS (C. Ingram-Smith et al., manuscript submitted). We have taken two approaches to investigate the mechanism of the novel AAS family. First, we have established a collaboration with Dr. Andrew Gulick (SUNY-Buffalo) to solve the structure of AAE. Crystals of the M. acetivorans AAS have been obtained and a 2.1 angstrom data set has been collected. A solved structure is anticipated in the next few months. Second, using amino sequence alignment with ACSs and otherproven short chain acyl-CoA synthetases, we are making targeted amino acid substitutions and biochemically and kinetically characterizing the resulting AAS variants to identify key amino acid residues in the mechanism.

Impacts
In addition to its metabolic role at the interface of a number of anabolic and catabolic pathways, ACS plays a critical role in linking metabolism to other physiological processes and may also play an important role in certain conditions such as prolonged fasting and diseases such as diabetes. Posttranslational regulation of ACS has been shown to be conserved in all three domains of life. In mammals, this enzyme is regulated by the sirtuins, which regulate metabolism at various points and are linked to aging. Surprisingly little is known about the biochemistry of this essential enzyme. We have identified key residues in ACS involved in substrate binding and catalysis and have identified a novel family of acyl-adenylate synthetases (AAS) related to the ACSs that catalyze the first step of the reaction but cannot catalyze the second step. I have a grant from NIH (GM069374-01A1) to investigate the active site of ACS and have recently submitted a proposal to NIH forcontinuation of this funding. I have also submitted a proposal to NSF to investigate the mechanism and physiological role of AAS. In the last year, two graduate student (two female including one rotation student) and six undergraduates (one female and one minority) have contributed to this project. A number of these students are authors on manuscripts that are in press, submitted, or in preparation. These students have gained valuable experience in biochemistry and molecular biology techniques as well as critical thinking skills that will serve them in their future endeavors in science or medicine.

Publications

Progress 01/01/05 to 12/31/05

Outputs
ACS, the principal enzyme for the conversion of acetate to acetyl-CoA, catalyzes a two step reaction that proceeds through an acetyl-AMP intermediate. An ACS (designated MT1) from the methane-producing thermophilic archaeon Methanobacterium thermoautotrophicus as our model enzyme. Four amino acid residues that form the binding pocket for acetate in MT1 have been identified and a manuscript detailing this characterization has been submitted. Several amino acid residues involved in either ATP or CoA binding and catalysis have been identified and characterized. The role of motif 3 in CoA synthetases has been unknown. We have shown that four of the residues comprising this motif are important for catalysis and Asp-502 is critical for proper binding of the ribose moiety of ATP. Two conserved Arg residues (Arg-528, Arg-586) have been shown to be vital for catalysis, while Arg-193 has been shown to be necessary for CoA binding and for catalysis An ACS-like enzyme in M.thermoautotrophicus, designated as MT3, has been shown to be an acyl-activating enzyme (AAE). This enzyme lacks the ability to complete the second step of the acyl-CoA synthetase reaction and instead releases the acyl-adenylate product from the first step. AAE hom*ologs have been identified in the completed genomes of almost all methanoarchaea and in a few anaerobic bacteria. AAE hom*ologs from M. thermoautotrophicus and Methanosarcina acetivorans have been biochemically and kinetically characterized. Both enzymes utilize ATP as the nucleotide triphosphate. Although both enzymes show the highest substrate affinity for 2-methyl butyrate, the preferred substrate for M. thermoautotrophicus AAE is butyrate and the preferred substrate for M. acetivorans AAE is propionate. The enzymes acetate kinase (AK) and phosphotransacetylase (PTA) form a pathway to activate acetate to acetyl-CoA. AK and PTA have been found only in prokaryotes from the Bacteria domain, with the exception of one genus fromthe Archaea domain. However, we identified AK in two phyla of fungi, the Ascomycota and the Basidiomycota. The absence of PTA in fungi suggests a new role for AK. We have identified a gene encoding D-xylulose 5-phosphate/D-fructose 6-phosphate phosphoketolase (XFP), a bacterial enzyme that utilizes acetyl phosphate as a substrate. This suggests that XFP and AK may function as a pathway in the fungi. This putative AK-XFP pathway is present in the basidiomycete Cryptococcus neoformans, a common invasive opportunistic pathogen of the central nervous system of AIDS patients and the most frequent cause of fungal meningitis worldwide. Genetic experiments indicate that the AK enzyme is essential for survival of C. neoformans. Interestingly, two XFP hom*ologs (designated XFP1 and XFP2) have been identified in the C. neoformans genome. Examination of the RNA expression levels of the AK and XFP genes indicate that XFP1 and AK are expressed under a similar growth condition. The cDNAs for AK andXFP1 have been cloned and the recombinant enzymes have been produced in E. coli. Purification of both enzymes for a complete biochemical and kinetic characterization is ongoing.

Impacts
Acetyl-CoA, an essential intermediate in metabolism, can be synthesized from acetate and CoA via three enzymatic pathways. One of these pathways, acetyl-CoA synthetase (ACS), is nearly ubiquitous in all living organisms. ACSs are closely related to other acyl-CoA synthetases that utilize short and medium chain acyl substrates. Many of these synthetases have important medical, agricultural, and biotechnological impact. The goal of the proposed research is to provide an in-depth understanding of ACS and related enzymes utilizing biochemical and kinetic approaches. The enzymes acetate kinase (AK) and phosphotransacetylase (PTA) form a second pathway to activate acetate to acetyl-CoA. AK and PTA have been found only in bacteria; however, we identified AK in a number of eukaryotic microbes, including two phyla of fungi. The absence of PTA in these fungi suggests a new role for AK. AK is thought to form a novel pathway with a phosphoketolase XFP which has been identified inall fungi that have AK. Preliminary experiments indicate that AK is essential in Cryptococcus neoformans, an opportunistic pathogen of the central nervous system of AIDS patients and the most frequent cause of fungal meningitis. Humans, animals, and plants lack AK and XFP; thus, their presence in pathogenic eukaryotic fungi may provide novel targets for anti-fungal agents. The proposed study has a broad educational impact at the graduate and undergraduate levels and provides a wealth of projects for students of all levels of ability. Both levels of students have made significant contributions to this project.

Publications

Progress 01/01/04 to 12/31/04

Outputs
Acetyl-CoA synthetase (ACS) catalyzes the conversion of acetate, ATP, and CoA into acetyl-CoA through an acetyl-adenylate intermediate. A structure-function approach has been taken to determine what amino acid residues in the Methanothermobacter thermoautotrophicus ACS (designated as MT1) are important for substrate binding and catalysis. Since the structure of the Salmonella enterica enzyme has been used as one foundation in our investigations of substrate binding in MT1, comparing the kinetic parameters would be useful to ensure that these enzymes have similar characteristics with respect to substrates. The Salmonella ACS shows a preference for acetate, although propionate can also be utilized with an 11.5-fold lower catalytic efficiency. Although the S. enterica ACS has a much lower turnover rate than MT1, the Km values for CoA and ATP are similar for both enzymes. The Salmonella ACS show a strong specificity for ATP as the nucleotide substrate and the requirementfor a divalent metal is absolute. Gel filtration indicates the S. enterica ACS is a monomer. The S. enterica ACS was crystallized in the presence of CoA and adenosine-5'-propylphosphate, an inhibitor that mimics the acetyl-adenylate intermediate. The propyl group of this inhibitor may approximate the position of acetate in the active site and is bound near a hydrophobic pocket formed by Ile-312, Thr-313, Val-388, and Trp-416. ACS is very closely related to the propionyl-CoA synthetase (PCS), which utilizes propionate as a substrate, and human SA and MACS, which utilize isobutyrate and octanoate, respectively. Alignment of ACS sequences with the PCS, SA, and MACS sequences reveals that the residues constituting the hydrophobic pocket in ACS have specific replacements in PCS, SA, and MACS. Some of the results for altering Val-388 to Ala and Trp-416 to Gly have been previously reported. Both variants have altered substrate specificities in which acetate is no longer the preferredsubstrate. Additional work on the W416G variant indicates that this alteration allows the enzyme to utilize substrates as long as octanoate as well as using branched chain substrates 2-methylvalerate, 3-methylvalerate, and 4-methylvalerate. These results indicate that Trp-416 is the prime determinant of the acyl substrate chain length that can be accommodated in the binding pocket. Ile-312 of ACS is replaced by Ala in PCS, SA, and MACS. The T313K MT1 variant has reduced affinity for both acetate and propionate and the turnover number is reduced two-fold. Thr-313 of ACS is replaced by Val in PCS and MACS and by Lys in SA. The T313K MT1 variant has poor affinity for both acetate and propionate. In contrast, the T313V variant has a significantly lower Km for both acetate and propionate than the wild type enzyme. Although the turnover rates for these two substrates are low, the catalytic efficiency of this variant with each substrate is very close to that of the wild type enzyme. Oneexplanation for this result is that the Val substitution at this position makes the pocket more hydrophobic, thus making it more habitable for acyl substrates.

Impacts
Acetyl-CoA, an essential intermediate at the junction of anabolic and catabolic pathways, can be synthesized from acetate and CoA via three enzymatic pathways. One of these pathways, acetyl-CoA synthetase (ACS), is nearly ubiquitous in all living organisms. ACSs are closely related to other acyl-CoA synthetases that utilize short and medium chain acyl substrates. Many of these synthetases have important medical, agricultural, and biotechnological impact. The goal of the proposed research is to provide an in-depth understanding of ACS and related enzymes utilizing biochemical and kinetic approaches. Using the structure of the Salmonella enterica ACS, residues in the acetate binding pocket of ACS and have shown that alteration of certain of these residues can greatly affect substrate utilization to allow much longer acyl substrates to be accommodated. The proposed study has a broad educational impact at the graduate and undergraduate levels and provides a wealth ofprojects for students of all levels of ability. Both levels of students have and are expected to continue to make significant contributions to this project. Our detailed investigation into the acetate binding site of ACSs will shed new light on the evolution of substrate specificity in the short and medium chain acyl-CoA synthetases.

Publications

Progress 01/01/03 to 12/31/03

Outputs
Recently, the structure was reported for the Salmonella enterica AMP-ACS bound to both CoA and adenosine-5'-propylphosphate, an inhibitor of the related propionyl-CoA synthetase that mimics the acetyl-adenylate intermediate. We hypothesize that the propyl group of adenosine-5'-propyl phosphate may mimic the location of acetate in the active site. Gulick et al. proposed that the propyl group is bound in a hydrophobic pocket formed by Val-386 and Trp-414. Val-386 (Val-388 of MT1) and Trp-414 (Trp-416 of MT1) are completely conserved among the AMP-ACS sequences. AMP-ACSs are very closely related to the propionyl-CoA synthetases (PCS), which utilize propionate as substrate instead of acetate, and human SA and MACS1, which utilize isobutyrate and octanoate, respectively. Alignment of AMP-ACS sequences with the PCS, SA, and MACS1 sequences reveals that Val-388 of MT1 is replaced by Ala in the PCS and SA and by Gly in MACS1. Trp-416 of MT1 is present in AMP-ACS and PCS butis replaced by Gly in SA and MACS1. Val-388 and Trp-416 of MT1 have been altered to Ala and Gly, respectively. The variants have been heterologously produced, purified and the kinetic parameters have been determined (Table 2). Table 2. Enzyme Substrate Km (mM) kcat(sec-1) kcat/Km (sec-1 mM-1) Wild Type acetate 2.9 +- 0.1 45.3 +- 0.3 10.7 +- 0.1 propionate 36.1 +- 1.2 36.3 +- 0.03 1.0 +- 0.03 V388A acetate 13.2 +- 0.6 36.2 +- 0.4 2.7 +- 0.1 propionate 4.1 +- 0.2 13.2 +- 0.1 3.2 +- 0.2 W416G acetate 132.1 +- 9.9 33.7 +- 0.8 0.26 +- 0.01 propionate 188.8 +- 1.2 38.5 +- 0.1 0.20 +- 0.001 butyrate 39.2 +- 0.2 21.6 +- 0.1 0.55 +- 0.01 valerate 19.8 +- 2.0 8.8 +- 0.4 0.09 +- 0.001 hexanoate 8.0 +- 0.2 1.6 +- 0.01 0.20 +- 0.005 Wild type MT1 shows activity with both acetate and propionate. Although the kcat values for both substrates are similar, the Km for acetate is 11-fold lower than that for propionate. As compared to the wild type enzyme, the Km for acetate for V388A variant is 4.6 foldhigher while that for propionate is 8.8 fold lower. The kcat for acetate for the variant is similar to that shown by the wild type, but the kcat for propionate is 2.7 fold lower. This single change at makes the variant a 3.2 fold more catalytically efficient enzyme with propionate than the wild type. The W416G variant shows altered substrate specificity and is now able to utilize butyrate, valerate, and hexanoate, substrates that the wild type cannot utilize. The Km for butyrate for the variant is in fact similar to the Km for propionate for the wild type MT1 . The kcat values for acetate and propionate for the W416G variant are not significantly affected and the kcat for butyrate is only two fold down from the kcat for acetate for the wild type enzyme Although the catalytic efficiency of this enzyme is low on all substrates, the efficiency observed with butyrate as the substrate is twice that observed with acetate or propionate, indicating that this enzyme now functions better as abutyryl-CoA synthetase. Our kinetic data for the V338A and W316G variants thus far suggest that both of these residues are part of the acetate binding pocket and primarily affect substrate binding but not catalysis.

Impacts
AMP-ACS is a member of a diverse superfamily of acyl-adenylate-forming enzymes that includes acyl-CoA ligases, enzymes that mediate the synthesis of peptide and polyketide secondary metabolites, firefly luciferase, and alpha-aminoadipate reductase. Many of these family members have important medical, agricultural, and biotechnological impact. The recent structure of the Salmonella enterica AMP-ACS now allows predictions to be made regarding the active site and residues important for substrate binding and catalysis. The goal of the proposed research is to provide an in-depth understanding of AMP-ACS and related enzymes utilizing biochemical and kinetic approaches. Our detailed investigation into the role of the active site of AMP-ACS and is expected to provide insight into understnding how this family of enzymes recognizes and interacts with its substrates.

Publications

Progress 01/01/02 to 12/31/02

Outputs
We have identified three ORFs in the genome of the methane-producing archaeon Methanobacterium thermoautotrophicum that could encode for AMP-forming acetyl-CoA synthetases (AMP-ACS). These genes, designated as MT1, MT2, and MT3 have been cloned into the pETBlue-1 vector for expression in Escherichia coli. The MT1 and MT3 proteins were produced as soluble, active enzymes; however, only MT1 has AMP-ACS activity. MT1 has been purified to hom*ogeneity by a three step chromatographic scheme. The enzyme shows a preference for acetate as the substrate, with little activity observed with propionate and no activity observed with longer chain substrates such as butyrate or branched chain substrates such as 2-methylpropionate. The kinetic parameters Km and kcat were determined for acetate (3.7 mM and 48.3 sec-1), MgATP (6.7 mM and 58.9 sec-1), and CoA (0.18 mM and 47.8 sec-1). Chemical modification has been extensively used as a means of studying the functional role(s) of aminoacid residues in proteins. Incubation of MT1 with the cysteine (Cys)-specific modifying reagent N-ethylmaleimide (NEM) resulted in time- and concentration dependent loss of enzymatic activity. A plot of pseudo-first order rate constant kobs values versus NEM concentration was linear, suggesting that at least one modifiable Cys residue is essential for activity. Incubation of MT1 with the arginine (Arg)-specific modifying reagent phenylglyoxal (PG) also resulted in time- and concentration dependent loss of enzymatic activity and a plot of kobs values versus PG concentration was linear, suggesting that at least one modifiable Arg residue is essential for activity. Substrate protection from inhibition was tested to examine whether the modified Cys and Arg residues responsible for inactivation reside in the active site. This protective effect delineates the extent to which modifications within the substrate binding site inhibit enzymatic activity. Acetate provided only slight protectionfrom NEM inactivation and CoA provided strong protection, whereas the level of protection provided by acetate + CoA was additive. MT1 precipitated in the presence of ATP or MgATP alone. However, the presence of acetate + MgATP not only prevented precipitation but also provided strong protection from inactivation by NEM. This may be due to formation of an acetyl-AMP enzyme intermediate that protects one or more active site Cys residues from NEM modification. Acetate offered little protection from PG, whereas CoA provided strong protection from PG at a level similar to that observed with NEM. Unlike the case observed for NEM, the protection provided by acetate + CoA was not additive but equaled that of CoA alone. Acetate + MgATP provided the strongest protection from PG inhibition, significantly higher than that observed for NEM. Again, the absence of protein precipitation and the protection provided by acetate + MgATP may be indicative of formation of an acetyl-AMP enzyme intermediateand/or a conformational change that prevents one or more Arg residues from being modified by PG.

Impacts
AMP-forming acetyl-CoA synthetase (AMP-ACS) is widespread in all organisms and is a key enzyme in metabolism. We have now determined that cysteine and arginine residues are important for the activity of AMP-ACS. Identification and characterization of the roles of these residues will contribute to a deeper understanding of the AMP-ACS enzyme family. The broader impacts of this proposal are several-fold. Graduate and undergraduate students will be involved in all aspects of this project. A minority student from Florida A&M pursuing his M.S. degree in Biochemistry is playing an integral role in this study. In addition, high school students of the South Carolina Governor's School will continue to participate in this project. All of the students involved in this project will be encouraged to present their research at the Clemson University Research Forum, state and regional scientific meetings, as well as national meetings. Collaborations with a laboratory at MIT has beenestablished for aspects of this project will broaden the expertise of the laboratory and add a new research area, thus offering new educational research opportunities for students at Clemson University. The biochemistry of AMP-ACSs will provide a basis for investigating their role in the physiology and metabolism of archaea, which play important roles in the production of greenhouse gases and souring of oil reservoirs. A better understanding the mechanism of AMP-ACS is expected to provide an insight into the mechanism of related proteins such as human Sa, which appears to be involved in hypertension.

Publications