Carbonic anhydrase (CA) plays important roles in biological processes such as photosynthesis, respiration, secretion of HCO3 -, pH homeostasis and ion exchange.The proteins commonly contain a zinc ion in the active site for catalyzing the hydration of CO2 and vice versa.It is known that there are three classes of CA, designated α-, βand γ-CAs, depending on the amino acid sequence similarities.The α-class is different from others in the structural architecture.Furthermore, even in the α-class, the enzyme from unicellular green alga, Chlamydomonas reinhardtii (chCA) is unique in posttranslational modifications that it is glycosylated and spliced into two peptides.Such glycosylations are found in only mammalian CAs but they are not spliced.To reveal the structural details and the role of N-glycosylation, an X-ray analysis of chCA has been performed.chCA is a homodimeric protein, the two subunits being crystallographically independent.In each subunit, residues from Ser298 to Asn345 are spliced to separate into long and short peptides.The two subunits are, however, linked together by a disulfide bond.In the catalytic site, a zinc ion is bound to the three conserved His163, His165 and His182 in a tetrahedral configuration.A water molecule is trapped at the fourth position of the Zn atom.The electron density maps indicate that N-glycosylations occur at the three sites, Asn101, Asn135 and Asn297.This structure is the first example of CA attached to N-glycosides.chCA molecules are interacted to each other with a six-fold screw symmetry to form a long column.Furthermore, they are fused through the lateral interactions like a beehive.Each catalytic site is exposed to the central tunnel.It suggests that chCA in the crystalline state also catalyze the reaction.
The biosynthesis of coenzyme B12 is a complex process involving more than two dozen enzymatic reactions. Near the end of this biosynthetic pathway cobyric acid synthetase (CbiP) catalyzes the remarkable amidation of four separate carboxylate groups within a single substrate, adenosyl-cobyrinic acid a,c-diamide. The time course for the multiple amidation reactions demonstrates that the partially amidated products are released from the active site after every round of catalysis, and thus the mechanism of the reaction is dissociative. The partially amidated intermediates were shown to be single chemical entities demonstrating that the four carboxylate groups are amidated in a specific reaction sequence. NMR spectroscopy was used to establish that carboxylate e was the first to be amidated followed in turn by d, b, and g. These results indicate that the initial substrate can productively bind to the enzyme active site in only one of four possible orientations. After the amidation of carboxylate e, the first partially amidated intermediate must dissociate from the active site and rebind in an orientation that is rotated by approximately 90°. Similar events must therefore follow after the amidation of carboxylates d and b. The structural basis for the dissociative and sequential reaction mechanism coupled with the rigid regiochemistry is unknown.
Cobyric acid synthetase (CbiP) from Salmonella typhimurium catalyzes the glutamine and ATP-dependent amidation of carboxylates b, d, e, and g within adenosyl cobyrinic acid a,c-diamide. After each round of catalysis the partially amidated intermediates are released into solution and the four carboxylates are amidated in the sequential order of e, d, b, and g for the wild type enzyme. In the presence of [γ-18O4]-ATP and adenosyl cobyrinic a,c-diamide the enzyme will catalyze the positional isotope exchange of the βγ-bridge oxygen with the two β-nonbridge oxygens. These results support the proposal that ATP is used to activate the carboxylate groups via the formation of a phosphorylated intermediate. CbiP catalyzes the hydrolysis of glutamine in the absence of ATP or adenosyl cobyrinic acid a,c-diamide, but the rate of glutamine hydrolysis is enhanced by a factor of 60 in the presence of these two substrates together. This result suggests that the formation of the phosphorylated intermediate is coupled to the activation of the site utilized for the hydrolysis of glutamine. However, the rate of glutamine hydrolysis is approximately 2.5 times the rate of ADP formation, indicating that the two active sites are partially uncoupled from one another and that some of the ammonia from glutamine hydrolysis leaks into the bulk solution. The mutation of D146 to either alanine or asparagine results in a protein that is able to catalyze the formation of cobyric acid. However, the strict amidation order observed with the wild type CbiP is partially randomized with carboxylate b being amidated last. With the D146N mutant, the predominant pathway occurs in the sequence d, e, g, and b. It is proposed that this residue enforces the amidation order in the wild type enzyme via charge−charge repulsion between the side chain carboxylate and the carboxylates of the substrate.
Journal Article Agglutinability of Mastitis Streptococci Get access W. N. Plastridge, W. N. Plastridge From the Department of Animal Diseases, Storrs Agricultural Experiment Station, Storrs, Connecticut Search for other works by this author on: Oxford Academic PubMed Google Scholar Laura F. Banfield, Laura F. Banfield From the Department of Animal Diseases, Storrs Agricultural Experiment Station, Storrs, Connecticut Search for other works by this author on: Oxford Academic PubMed Google Scholar L. F. Williams L. F. Williams From the Department of Animal Diseases, Storrs Agricultural Experiment Station, Storrs, Connecticut Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Infectious Diseases, Volume 66, Issue 3, May 1940, Pages 202–211, https://doi.org/10.1093/infdis/66.3.202 Published: 01 May 1940 Article history Received: 01 February 1940 Published: 01 May 1940
