In this project the aim has been to study the model protein carbonic anhydrase in Neisseria gonorrhoeae, a bacterium whose carbonic anhydrase has great similarities both structurally and functionally with the human form. By measuring and comparing the wild type of NGCA with mutants lacking one of the four tryptophan residues it can be seen what effect these tryptophans has on stability and activity and then compare with the known data of HCA II to learn more about their differences and similarities. The results from the stability and activity measurements are that the wild type is by far the most stable protein with W141L mutant coming thereafter. From Trp-fluorescence and CO2-hydration measurement a clear two-transition steps (N→ I→ U) can be seen. This differs from earlier data where it instead only was a one-transition step for the wild type (N→U). The data is also very reliable and gives in most cases a perfect fit to the line. We also see this two-transition step for the other mutants stable enough, strengthening the theory further. One fact that could be drawn from all the measurements is that when an intermediate is formed the ability for the enzyme NGCA to perform it’s catalytically ability is disabled. Another thing is that the purification scheme of HCA II is not optimal to be directly applied to NGCA, despite the similarity in secondary and tertiary structure.

Analysis of the genome of Anabaena sp. PCC7120 reveals that it lacks the gene, ccaA, which encodes the bonafide carboxysomal, β-class carbonic anhydrase (CA) CcaA. However, the carboxysome enriched fraction of Anabaena PCC7120 exhibits CA activity. Bioinformatic analysis reveals that the N-terminal region of the carboxysome protein CcmM has high sequence and structural similarity to the γ-class CA of Methanosarcina thermophila. Recombinantly expressed CcmM is found to be inactive in in-vitro CA assays. E. coli cell extracts containing an overexpressed form of CcmM comprised of the N-terminal 209 amino acids (CcmM209) are also inactive. However, CcmM209 displays CA activity after incubation with the thiol oxidizing agent diamide or when bound to an affinity matrix. It appears that CcmM is indeed a functional γ-CA which is active under oxidizing condition. It is hypothesized that the C-terminal RbcS like domain in CcmM may regulate activity by allowing CcmM activation only when sequestered within the carboxysome.

Analysis of the genome of Anabaena sp. PCC7120 reveals that it lacks the gene, ccaA, which encodes the bonafide carboxysomal, β-class carbonic anhydrase (CA) CcaA. However, the carboxysome enriched fraction of Anabaena PCC7120 exhibits CA activity. Bioinformatic analysis reveals that the N-terminal region of the carboxysome protein CcmM has high sequence and structural similarity to the γ-class CA of Methanosarcina thermophila. Recombinantly expressed CcmM is found to be inactive in in-vitro CA assays. E. coli cell extracts containing an overexpressed form of CcmM comprised of the N-terminal 209 amino acids (CcmM209) are also inactive. However, CcmM209 displays CA activity after incubation with the thiol oxidizing agent diamide or when bound to an affinity matrix. It appears that CcmM is indeed a functional γ-CA which is active under oxidizing condition. It is hypothesized that the C-terminal RbcS like domain in CcmM may regulate activity by allowing CcmM activation only when sequestered within the carboxysome.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 73-77).

Thesis (Ph. D.)--Institute of Paper Chemistry, 1958. / Includes bibliographical references (p. 87-90).

No description available.

I denna avhandling presenteras arbete utfört med enzymet humant karboanhydras II (HCAII). Enzymer är en typ av proteiner som accelererar (katalyserar) kemiska reaktioner, vilket är nödvändigt för allt levande. Den naturliga funktionen för HCAII är att katalysera omvandlingen av gasen koldioxid till vätekarbonat, som är löslig i vätska. Detta är viktigt bl.a. för att koldioxid som bildas i kroppen, och fraktas i blodet i form av vätekarbonat, skall hinna över till utandningsluften under den korta tid blodet är i lungorna. Proteiner består av aminosyror som länkats samman i en lång kedja, där varje aminosyra är en av de 20 naturliga aminosyratyperna. Ett proteins struktur och egenskaper bestäms av aminosyrasekvensen, som i sin tur bestäms av genen för just det proteinet. Med genteknik kan ett proteins gen ändras (muteras), så att aminosyrasekvensen ändras, och det har här utnyttjats för att förändra HCAIIs katalytiska egenskaper. Förutom dess naturliga funktion kan HCAII även klyva (hydrolysera) vissa estrar. Mutationer gjordes så att en ’ficka’ i HCAIIs struktur, där molekylerna (substraten) som skall klyvas binder, fick en större volym. På så sätt skapades varianter med en kraftigt ökad kapacitet för att hydrolysera långa estersubstrat jämfört med icke-muterat HCAII. Som en utveckling av detta projekt skapades en mutant av HCAII, som kan hydrolysera ett än mer skrymmande substrat. I ett annat projekt har en ny katalytisk aktivitet skapats i HCAII, som inte utnyttjar enzymets naturliga katalytiska förmåga. Ett nytt estersubstrat konstruerades, med en del som binder kraftigt till HCAII, så att en stark substratbindning erhölls. Sedan muterades vissa aminosyror till en reaktiv aminosyra som heter histidin. Valet av positioner för mutation baserades på en datormodell av enzymet med bundet substrat. Eftersom histidin kan delta i hydrolysreaktioner, får det muterade enzymet möjlighet att klyva substratet. Flera olika mutanter testades, och den effektivaste innehöll ett nära kopplat par av histidiner. Denna mutant undersöktes mer noggrannt, vilket gav viss information om den katalytiska mekanismen. Det långsiktiga målet med detta arbete är att konstruera muterade enzymer som kan klyva giftiga ämnen, eller användas vid framställning av kemikalier. Det finns behov av nya enzymer för olika typer av substrat, och att med rationella metoder skapa nya katalytiska aktiviteter i proteiner är ett svårt vetenskapligt problem som ännu är i ett tidigt utvecklingsskede. / The main part of this thesis describes results from protein engineering experiments, in which the catalytic activity of the enzyme human carbonic anhydrase II (HCAII) is engineered by mutagenesis. This enzyme, which catalyzes the interconversion between CO2 and HCO3- in the body, also has the ability to hydrolyze ester bonds. In one project, the specificity of HCAII towards a panel of para-nitrophenyl ester substrates, with acyl chain lengths ranging from one to five carbon atoms, was changed by enlarging the substrate binding hydrophobic pocket. A variant was identified that has highly increased specificity towards substrates with long acyl chains. The mutant V121A/V143A hydrolyzes pNPV, which has four carbon atoms in the acyl chain, with an efficiency that is increased by a factor of 3000 compared to HCAII. Further, transition state analogues (TSAs) were docked to HCAII and mutant variants, and the results were correlated to the results from kinetic measurements. This indicated that automated docking could be used to some extent to construct HCAII variants with a designed specificity. Using this approach, a HCAII mutant that can hydrolyze a model benzoate ester was created. Interestingly, the resulting variant V121A/V143A/T200A was found to be highly active with other ester substrates as well. For pNPA, a kcat/KM of 1*105 M-1s-1 was achieved, which is the highest efficiency for hydrolysis of carboxylic acid esters reported for any HCAII variant. In another project, the strong affinity between the active site zinc ion and sulfonamide was used to achieve binding of a designed substrate. Thus, the natural Zn-OH- site of HCAII was not used for catalysis, but for substrate binding. The substrate contains a benzenesulfonamide part in one end, with a para-nitrophenyl ester connected via a linker. The linker was chosen to ensure that the scissile bond is positioned close to His-64 and histidine residues introduced by mutagenesis in other positions. Using this approach, an enzyme was designed with a distinctly new two-histidine catalytic site for ester hydrolysis. The mutant, F131H/V135H, has a kcat/KM of approximately 14000 M-1s-1, which corresponds to a rate enhancement of 107 compared to a histidine mimic. Finally, results are reported on a project aimed at cloning and producing a putative carbonic anhydrase from the malaria parasite Plasmodium falciparum. The gene was cloned by PCR and the construct was overexpressed in E. coli. However, the resulting protein was not soluble, and initial attempts to refold it are also reported.

Thesis (Ph. D.)--University of California, San Diego, 1998. / Vita. Includes bibliographical references (p. 150-155).

Thesis (doctoral)--Chalmers Tekniska högskola, 1972. / "Akademisk avhandling för filosofie doktorsexamen i kemi ... fredagen den 2 juni 1972 ... Chalmers tekniska högskola."

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 116 pages. Includes Vita. Includes bibliographical references.