Modulation of activity and substrate binding modes by mutation of single and double subsites +1/+2 and -5/-6 of barley alpha-amylase 1.
Enzymatic properties of barley alpha-amylase 1 (AMY1) are altered as a result of amino acid substitutions at subsites -5/-6 (Cys95-->Ala/Thr) and +1/+2 (Met298-->Ala/Asn/Ser) as well as in the double mutants, Cys95-->Ala/Met298-->Ala/Asn/Ser. Cys95-->Ala shows 176% activity towards insoluble Blue Starch compared to wild-type AMY1, kcat of 142 and 211% towards amylose DP17 and 2-chloro-4-nitrophenyl beta-d-maltoheptaoside (Cl-PNPG7), respectively, but fivefold to 20-fold higher Km. The Cys95-->Thr-AMY1 AMY2 isozyme mimic exhibits the intermediary behaviour of Cys95-->Ala and wild-type. Met298-->Ala/Asn/Ser have slightly higher to slightly lower activity for starch and amylose, whereas kcat and kcat/Km for Cl-PNPG7 are < or = 30% and < or = 10% of wild-type, respectively. The activity of Cys95-->Ala/Met298-->Ala/Asn/Ser is 100-180% towards starch, and the kcat/Km is 15-30%, and 0.4-1.1% towards amylose and Cl-PNPG7, respectively, emphasizing the strong impact of the Cys95-->Ala mutation on activity. The mutants therefore prefer the longer substrates and the specificity ratios of starch/Cl-PNPG7 and amylose/Cl-PNPG7 are 2.8- to 270-fold and 1.2- to 60-fold larger, respectively, than of wild-type. Bond cleavage analyses show that Cys95 and Met298 mutations weaken malto-oligosaccharide binding near subsites -5 and +2, respectively. In the crystal structure Met298 CE and SD (i.e., the side chain methyl group and sulfur atom) are near C(6) and O(6) of the rings of the inhibitor acarbose at subsites +1 and +2, respectively, and Met298 mutants prefer amylose for glycogen, which is hydrolysed with a slightly lower activity than by wild-type. Met298 AMY1 mutants and wild-type release glucose from the nonreducing end of the main-chain of 6"'-maltotriosyl-maltohexaose thus covering subsites -1 to +5, while productive binding of unbranched substrate involves subsites -3 to +3.
Mori
H
H
Carlsberg Laboratory, Department of Chemistry, Gamle Carlsberg, Copenhagen Valby, Denmark.
Bak-Jensen
K S
KS
Gottschalk
T E
TE
Motawia
M S
MS
Damager
I
I
Møller
B L
BL
Svensson
B
B
D000688:Amylose / Q000378:metabolism; D001483:Base Sequence; D017931:DNA Primers; D006003:Glycogen / Q000378:metabolism; D001467:Hordeum / Q000201:enzymology*; D006868:Hydrolysis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D017931:DNA Primers; D009844:Oligosaccharides; C021705:maltooligosaccharides; D006003:Glycogen; D000688:Amylose; D000516:alpha-Amylases