1kbb: Difference between revisions

Revision as of 14:32, 21 February 2008

Mechanistic Analyses of Catalysis in Human Pancreatic alpha-Amylase: Detailed Kinetic and Structural Studies of Mutants of Three Conserved Carboxylic Acids

OverviewOverview

The roles of three conserved active site carboxylic acids (D197, E233, and D300) in the catalytic mechanism of human pancreatic alpha-amylase (HPA) were studied by utilizing site-directed mutagenesis in combination with structural and kinetic analyses of the resultant enzymes. All three residues were mutated to both alanine and the respective amide, and a double alanine mutant (E233A/D300A) was also generated. Structural analyses demonstrated that there were no significant differences in global fold for the mutant enzymes. Kinetic analyses were performed on the mutants, utilizing a range of substrates. All results suggested that D197 was the nucleophile, as virtually all activity (>10(5)-fold decrease in k(cat) values) was lost for the enzymes mutated at this position when assayed with several substrates. The significantly greater second-order rate constant of E233 mutants on "activated" substrates (k(cat)/K(m) value for alpha-maltotriosyl fluoride = 15 s(-)(1) mM(-)(1)) compared with "unactivated" substrates (k(cat)/K(m) value for maltopentaose = 0.0030 s(-)(1) mM(-)(1)) strongly suggested that E233 is the general acid catalyst, as did the pH-activity profiles. Transglycosylation was favored over hydrolysis for the reactions of several of the enzymes mutated at D300. At the least, this suggests an overall impairment of the catalytic mechanism where the reaction then proceeds using the better acceptor (oligosaccharide instead of water). This may also suggest that D300 plays a crucial role in enzymic interactions with the nucleophilic water during the hydrolysis of the glycosidic bond.

About this StructureAbout this Structure

1KBB is a Single protein structure of sequence from Homo sapiens with and as ligands. Active as Alpha-amylase, with EC number 3.2.1.1 Full crystallographic information is available from OCA.

ReferenceReference

Mechanistic analyses of catalysis in human pancreatic alpha-amylase: detailed kinetic and structural studies of mutants of three conserved carboxylic acids., Rydberg EH, Li C, Maurus R, Overall CM, Brayer GD, Withers SG, Biochemistry. 2002 Apr 2;41(13):4492-502. PMID:11914097

Page seeded by OCA on Thu Feb 21 13:32:17 2008

Proteopedia Page Contributors and Editors (what is this?)Proteopedia Page Contributors and Editors (what is this?)

OCA

@@ Line 1: / Line 1: @@
-[[Image:1kbb.gif|left|200px]]<br />
+[[Image:1kbb.gif|left|200px]]<br /><applet load="1kbb" size="350" color="white" frame="true" align="right" spinBox="true"
-<applet load="1kbb" size="450" color="white" frame="true" align="right" spinBox="true"
 caption="1kbb, resolution 1.9&Aring;" />
 '''Mechanistic Analyses of Catalysis in Human Pancreatic alpha-Amylase: Detailed Kinetic and Structural Studies of Mutants of Three Conserved Carboxylic Acids'''<br />
 ==Overview==
-The roles of three conserved active site carboxylic acids (D197, E233, and, D300) in the catalytic mechanism of human pancreatic alpha-amylase (HPA), were studied by utilizing site-directed mutagenesis in combination with, structural and kinetic analyses of the resultant enzymes. All three, residues were mutated to both alanine and the respective amide, and a, double alanine mutant (E233A/D300A) was also generated. Structural, analyses demonstrated that there were no significant differences in global, fold for the mutant enzymes. Kinetic analyses were performed on the, mutants, utilizing a range of substrates. All results suggested that D197, was the nucleophile, as virtually all activity (&gt;10(5)-fold decrease in, k(cat) values) was lost for the enzymes mutated at this position when, assayed with several substrates. The significantly greater second-order, rate constant of E233 mutants on "activated" substrates (k(cat)/K(m) value, for alpha-maltotriosyl fluoride = 15 s(-)(1) mM(-)(1)) compared with, "unactivated" substrates (k(cat)/K(m) value for maltopentaose = 0.0030, s(-)(1) mM(-)(1)) strongly suggested that E233 is the general acid, catalyst, as did the pH-activity profiles. Transglycosylation was favored, over hydrolysis for the reactions of several of the enzymes mutated at, D300. At the least, this suggests an overall impairment of the catalytic, mechanism where the reaction then proceeds using the better acceptor, (oligosaccharide instead of water). This may also suggest that D300 plays, a crucial role in enzymic interactions with the nucleophilic water during, the hydrolysis of the glycosidic bond.
+The roles of three conserved active site carboxylic acids (D197, E233, and D300) in the catalytic mechanism of human pancreatic alpha-amylase (HPA) were studied by utilizing site-directed mutagenesis in combination with structural and kinetic analyses of the resultant enzymes. All three residues were mutated to both alanine and the respective amide, and a double alanine mutant (E233A/D300A) was also generated. Structural analyses demonstrated that there were no significant differences in global fold for the mutant enzymes. Kinetic analyses were performed on the mutants, utilizing a range of substrates. All results suggested that D197 was the nucleophile, as virtually all activity (&gt;10(5)-fold decrease in k(cat) values) was lost for the enzymes mutated at this position when assayed with several substrates. The significantly greater second-order rate constant of E233 mutants on "activated" substrates (k(cat)/K(m) value for alpha-maltotriosyl fluoride = 15 s(-)(1) mM(-)(1)) compared with "unactivated" substrates (k(cat)/K(m) value for maltopentaose = 0.0030 s(-)(1) mM(-)(1)) strongly suggested that E233 is the general acid catalyst, as did the pH-activity profiles. Transglycosylation was favored over hydrolysis for the reactions of several of the enzymes mutated at D300. At the least, this suggests an overall impairment of the catalytic mechanism where the reaction then proceeds using the better acceptor (oligosaccharide instead of water). This may also suggest that D300 plays a crucial role in enzymic interactions with the nucleophilic water during the hydrolysis of the glycosidic bond.
 ==About this Structure==
-KBB is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with CA and CL as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Alpha-amylase Alpha-amylase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.1 3.2.1.1] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1KBB OCA].
+KBB is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with <scene name='pdbligand=CA:'>CA</scene> and <scene name='pdbligand=CL:'>CL</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Alpha-amylase Alpha-amylase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.1 3.2.1.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1KBB OCA].
 ==Reference==
@@ Line 15: / Line 14: @@
 [[Category: Homo sapiens]]
 [[Category: Single protein]]
-[[Category: Brayer, G.D.]]
+[[Category: Brayer, G D.]]
 [[Category: Li, C.]]
 [[Category: Maurus, R.]]
-[[Category: Overall, C.M.]]
+[[Category: Overall, C M.]]
-[[Category: Rydberg, E.H.]]
+[[Category: Rydberg, E H.]]
-[[Category: Withers, S.G.]]
+[[Category: Withers, S G.]]
 [[Category: CA]]
 [[Category: CL]]
@@ Line 35: / Line 34: @@
 [[Category: pancreatic]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Mon Nov 12 17:49:35 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:32:17 2008''