Dear Rathinam,

This kind of enzyme is very specific. It can act on p-nitrophenyl-beta-galactosidase but not on the alpha isomer.

β-galactosidase,  β-gal, is a glycoside hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides through the breaking of a glycosidic bond. β-galactosides include carbohydrates containing galactose where the glycosidic bond lies above the galactose molecule. Substrates of different β-galactosidases include ganglioside GM1, lactosylceramides, lactose, and various glycoproteins. β-galactosidase can catalyze two different reactions in organisms. In one, it can go through a process called transgalactosylation to make allolactose, creating a positive feedback loop for the production of β-gal. It can also hydrolyze lactose into galactose and glucose which will proceed into glycolysis. The active site of β-galactosidase catalyzes the hydrolysis of its disaccharide substrate via "shallow" (nonproductive site) and "deep" (productive site) binding. Galactosides such as PETG and IPTG will bind in the shallow site when the enzyme is in "open" conformation while transition state analogs such as L-ribose and D-galactonolactone will bind in the deep site when the conformation is "closed". The enzymatic reaction consists of two chemical steps, galactosylation (k2) and degalactosylation (k3). Galactosylation is the first chemical step in the reaction where Glu461 donates a proton to a glycosidic oxygen, resulting in galactose covalently bonding with Glu537. In the second step, degalactosylation, the covalent bond is broken when Glu461 accepts a proton, replacing the galactose with water. Two transition states occur in the deep site of the enzyme during the reaction, once after each step. When water participates in the reaction, galactose is formed, otherwise, when D-glucose acts as the acceptor in the second step, transgalactosylation occurs . It has been kinetically measured that single tetramers of the protein catalyze reactions at a rate of 38,500 ± 900 reactions per minute.[9] Monovalent potassium ions (K+) as well as divalent magnesium ions (Mg2+) are required for the enzyme's optimal activity. The beta-linkage of the substrate is cleaved by a terminal carboxyl group on the side chain of a glutamic acid. In E. coli, Glu-461 was thought to be the nucleophile in the substitution reaction. However, it is now known that Glu-461 is an acid catalyst. Instead, Glu-537 is the actual nucleophile, binding to a galactosyl intermediate. In humans, the nucleophile of the hydrolysis reaction is Glu-268. Gly794 is important for β-gal activity. It is responsible for putting the enzyme in an "closed", ligand bounded, conformation or "open" conformation, acting like a "hinge" for the active site loop. The different conformations ensure that only preferential binding occurs in the active site. In the presence of a slow substrate, Gly794 activity increased as well as an increase in galactosylation and decrease in degalactosylation.

Juers, Douglas H.; Hakda, Shamina; Matthews, Brian W.; Huber, Reuben E. (2003-11-01). "Structural Basis for the Altered Activity of Gly794 Variants of Escherichia coli β-Galactosidase†". Biochemistry 42 (46): 13505–13511. doi:10.1021/bi035506j. ISSN 0006-2960.

Juers, Douglas H.; Matthews, Brian W.; Huber, Reuben E. (2012-12-01). "LacZ β-galactosidase: Structure and function of an enzyme of historical and molecular biological importance". Protein Science 21 (12): 1792–1807. doi:10.1002/pro.2165. ISSN 1469-896X. PMC 3575911. PMID 23011886.

Gebler JC, Aebersold R, Withers SG (June 1992). "Glu-537, not Glu-461, is the nucleophile in the active site of (lac Z) beta-galactosidase from Escherichia coli". J. Biol. Chem. 267 (16): 11126–30. PMID 1350782.

Yuan J, Martinez-Bilbao M, Huber RE (April 1994). "Substitutions for Glu-537 of beta-galactosidase from Escherichia coli cause large decreases in catalytic activity". Biochem. J. 299 (Pt 2): 527–31. PMC 1138303. PMID 7909660.

McCarter JD, Burgoyne DL, Miao S, Zhang S, Callahan JW, Withers SG (January 1997). "Identification of Glu-268 as the catalytic nucleophile of human lysosomal beta-galactosidase precursor by mass spectrometry". J. Biol. Chem. 272 (1): 396–400. doi:10.1074/jbc.272.1.396. PMID 8995274.

The following is a recent review on this enzyme entitled "LacZ β-galactosidase: Structure and function of an enzyme of historical and molecular biological importance " which can shed light on its specifity:

Protein Sci. 2012 Dec; 21(12): 1792–1807.

Published online 2012 Sep 25. doi:  10.1002/pro.2165

PMCID: PMC3575911

LacZ β-galactosidase: Structure and function of an enzyme of historical and molecular biological importance

Douglas H Juers,1,* Brian W Matthews,2,* and Reuben E Huber3,*

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This article has been cited by other articles in PMC.

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Abstract

This review provides an overview of the structure, function, and catalytic mechanism of lacZ β-galactosidase. The protein played a central role in Jacob and Monod's development of the operon model for the regulation of gene expression. Determination of the crystal structure made it possible to understand why deletion of certain residues toward the amino-terminus not only caused the full enzyme tetramer to dissociate into dimers but also abolished activity. It was also possible to rationalize α-complementation, in which addition to the inactive dimers of peptides containing the “missing” N-terminal residues restored catalytic activity. The enzyme is well known to signal its presence by hydrolyzing X-gal to produce a blue product. That this reaction takes place in crystals of the protein confirms that the X-ray structure represents an active conformation. Individual tetramers of β-galactosidase have been measured to catalyze 38,500 ± 900 reactions per minute. Extensive kinetic, biochemical, mutagenic, and crystallographic analyses have made it possible to develop a presumed mechanism of action. Substrate initially binds near the top of the active site but then moves deeper for reaction. The first catalytic step (called galactosylation) is a nucleophilic displacement by Glu537 to form a covalent bond with galactose. This is initiated by proton donation by Glu461. The second displacement (degalactosylation) by water or an acceptor is initiated by proton abstraction by Glu461. Both of these displacements occur via planar oxocarbenium ion-like transition states. The acceptor reaction with glucose is important for the formation of allolactose, the natural inducer of the lac operon.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3575911/

Hoping this will be helpful,

Rafik

Hi there,

If the name of the enzyme includes the anomeric form of the galactose part of the substrate, it tends to prove the hydrolase activity is very specific of this kind of glycosidic bond involving the carbon 1 of galactose. As a general rule this enzyme is highly specific of the galactose part and much less specific on the rest which is why ONPG may be used for beta-galactosidase assay though it's structurally far from the glucose part of lactose.

I looked in BRENDA for references to the use of alpha-galactosides as substrates for beta-galactosidase.

http://brenda-enzymes.org/enzyme.php?ecno=3.2.1.23#SUBSTRATE

There are a few such references. This one for example indicates that the activity towards the alpha substrate is about 1% that towards the beta.

http://brenda-enzymes.org/literature.php?e=3.2.1.23&r=704135]

However, one must keep in mind the possibility that this was due to contamination with alpha-galactosidase.

Thankyou all.

@Rafik Karaman,

I downvoted your "answer" here and (there) because it is not a proper answer as it doesn't reply clearly to the question at all (no mention of any activity towards alpha anomer anywhere in it). Second you don't need a full lecture to answer a rather simple question. When I'm posting a question here, I surely do not wish any reply like yours and would prefer a short and accurate one on the issue raised by the question (and I make the guess that most of the users here are looking for simple and straight answers or advices for their scientific issues). And in my opinion copy and paste is not a scientific approach for a distinguished professor to spread the scientific knowledge (as basically anyone could do the same with a simple internet access). And btw why did you downvote my answer (because I guess it's you, isn't it)? Does it contain any mistake or improper explanation to this enzyme specificity? Thanks for in advance for your explanation...  

Dear Dominique Liger

I am not sure whether it is right or not to write here. But I cant agree with you about your statement that most of the users are looking for a simple and straight answer. For the expert like you, probably it is right. But researcher like me, it is very important sometimes to get a detailed answer. I think there are lots of users who are seeking a detailed answer. By the way, we are learning from you people having in depth research knowledge. I think we should have the mutual respect to each other along with scientific knowledge.

Dear Rafik,

I'm not teaching anything to anyone, just answering your request about my motivation as you wished... There is nothing disrespectful in my answer and especially nothing in line with your science achievement, financial support, religion or else... If you think there is, I'm very sorry.

Dear Rafik,

I am sure that Dominique is sincere in his response. A too strong a reaction is sometimes a matter of pride, sometimes a lack of self-confidence, but in my opinion, never a constructive attitude.

Regards

Dear all,

I tend to believe that Dominique did not mean to heart me and I consider the issue is closed.

I wish him and all of you a happy holiday,

Rafik